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In vitro antifungal activity of Ocimum selloi essential oil and methylchavicol against phytopathogenic fungi

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The efficacy of Ocimum selloi essential oil was evaluated for controlling the growth of mycelia and spores germination Moniliophthora perniciosa. Six compounds (99.89%) of the total oil were identified by GC-MS, of which methyl chavicol, methyl eugenol,-caryophyllene, germacrene-D, bicyclogermacrene and spathulenol. Essential oil was tested for anti-fungal activity, which was determined by disc diffusion and minimum inhibitory concentration (MIC) determination methods. Application of the oil reduced mycelial growth in a dose dependent manner, with maximum inhibition being observed at concentration of 1,000 ppm. Such antifungal activity could be attributed to methyl chavicol since the pure compound was shown to be similarly effective against Moniliophthora perniciosa at 1,000 ppm. The oil when applied at a concentration of 1,000 ppm, reduced the spore germination of Colletotrichum gloeosporioides and M. perniciosa by 93 and 87%, respectively, but had no effect on the Alternaria alternata. It is concluded that the oil from O. selloi and its major constituent, methyl chavicol, are efficient in inhibiting M. perniciosa, but less effective against C. gloeosporioides and A. alternata. The results obtained from this work may contribute to the development of alternative anti-fungal agents to protect the cacao crop from fungal disease.
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Revista Ciência Agronômica, v. 46, n. 2, p. 428-435, abr-jun, 2015
Centro de Ciências Agrárias - Universidade Federal do Ceará, Fortaleza, CE
www.ccarevista.ufc.br ISSN 1806-6690
Artigo Científico
In vitro antifungal activity of Ocimum selloi essential oil and
methylchavicol against phytopathogenic fungi1
Atividade antifúngica in vitro do óleo essencial de Ocimum selloi e metilchavicol
contra fungos fitopatogênicos
Larissa Corrêa Bomfim Costa2, José Eduardo Brasil Pereira Pinto3*, Suzan Kelly Vilela Bertolucci4, João Cássia
do Bomfim Costa5, Péricles Barreto Alves6 e Edenilson dos Santos Niculau6
ABSTRACT - The efficacy of Ocimum selloi essential oil was evaluated for controlling the growth of mycelia and spores
germination Moniliophthora perniciosa. Six compounds (99.89%) of the total oil were identified by GC-MS, of which methyl
chavicol, methyl eugenol, -caryophyllene, germacrene-D, bicyclogermacrene and spathulenol. Essential oil was tested for anti-
fungal activity, which was determined by disc diffusion and minimum inhibitory concentration (MIC) determination methods.
Application of the oil reduced mycelial growth in a dose dependent manner, with maximum inhibition being observed at
concentration of 1,000 ppm. Such antifungal activity could be attributed to methyl chavicol since the pure compound was shown
to be similarly effective against Moniliophthora perniciosa at 1,000 ppm. The oil when applied at a concentration of 1,000 ppm,
reduced the spore germination of Colletotrichum gloeosporioides and M. perniciosa by 93 and 87%, respectively, but had no
effect on the Alternaria alternata. It is concluded that the oil from O. selloi and its major constituent, methyl chavicol, are
efficient in inhibiting M. perniciosa, but less effective against C. gloeosporioides and A. alternata. The results obtained from this
work may contribute to the development of alternative anti-fungal agents to protect the cacao crop from fungal disease.
Key words: Antifungal Activity. Methyl Chavicol. Moniliophthora perniciosa. Theobroma cacao.
RESUMO - A eficácia do óleo essencial de Ocimum selloi na inibição do crescimento micelial e na germinação de esporos foi avaliada
in vitro para os fungos Moniliophthora perniciosa, Colletotrichum gloesporioides e Alternaria alternata. Seis constituintes (99,89%)
do total do óleo foram identificados por CG-EM metil chavicol, metil eugenol, -cariofileno, germacreno-D, biclogermacreno
e espatulenol. O óleo essencial foi testado para atividade antifúngica, o qual foi determinado pelos métodos de difusão e
concentração mínima inibitória (MIC). A aplicação do óleo reduziu o crescimento micelial dependente da dose, com máxima
inibição na concentração de 1.000 ppm. A atividade antifúngica pode ser atribuída ao composto puro metil chavicol. Este
mostrou ser efetivo contra M. perniciosa em 1.000 ppm. O óleo quando aplicado na concentração de 1.000 ppm, reduziu
a germinação do esporo de Colletotrichum gloeosporioides e M. perniciosa em 93 e 87%, respectivamente, mas não teve
efeito em Alternaria alternata. Conclui-se que o óleo essencial e seu constituinte majoritário, metil chavicol, são eficientes na
inibição de M. perniciosa, mas menos efetivo contra C. gloeosporioides e A. alternata. Os resultados obtidos nesta pesquisa
pode contribuir para o desenvolvimento de um agente alternativo para proteger a cultura do cacao contra esta doença fúngica.
Palavras-chave: Atividade Antifúngica. Metil chavicol. Moniliophthora perniciosa, Theobroma cacao.
Autor para correspondência
1Recebido para publicação em 08/11/2013; aprovado em 16/01/2015
Parte da Tese de Doutorado do primeiro autor apresentada na Universidade Federal de Lavras/UFLA
2Departamento de Ciência Biológica/Anatomia e Plantas Medicinais, Universidade Estadual de Santa Cruz, Ilhéus-BA, Brasil, larissa@uesc.br
3Departamento de Agricultura/Cultura de Tecidos e Plantas Medicinais, Universidade Federal de Lavras, Av. Doutor Sylvio Menicucci, 1001,
Kennedy, Câmpus Universitário, Caixa Postal 3037, Lavras-MG, Brasil, 37.200-000, jeduardo@dag.ufla.br
4Departamento de Agricultura/Fitoquimica e Plantas Medicinais, Universidade Federal de Lavras, Lavras-MG, Brasil, suzan@dag.ufla.br
5Centro de Pesquisas do Cacau, Itabuna-BA, Brasil, jcbcosta@uol.com.br
6Departamento de Química, Universidade Federal de Sergipe, São Cristóvão-SE, Brasil, periclesbalves@gmail.com, edenilsonnicolau@hotmail.com
Rev. Ciênc. Agron., v. 46, n. 2, p. 428-435, abr-jun, 2015 429
L. C. B. Costa et al.
INTRODUCTION
The essential oils of numerous aromatic and
medicinal plants are known to possess antimicrobial
activity (CAROVI-STANKO et al., 2010). Indeed,
a wide range of plant secondary metabolites exhibit
antimicrobial properties, and these are often associated
with the natural defense mechanism against attack
by phytopathogenic bacteria and fungi (JANSSEN;
SCHEFFER; BAERHEIM-SVENDSEN, 1987). In
this context, a number of studies have shown that
crude plant extracts and essential oils are potentially
useful for controlling various types of phytopathogens
(CARVALHO et al., 2013; RAHMAN; HOSSAIN;
KANG, 2010; SILVA et al., 2012a; SILVA et al., 2012b;
SILVA; BASTOS 2007; VIGO-SCHULTZ et al., 2006).
Alternaria alternata (Fr.) Keissl., Colletotrichum
spp. and Moniliophthora perniciosa (Stahel) Aime &
Phillips-Mora [syn. Crinipellis perniciosa (Stahel) Singer]
are representative of phytopathogens that cause significant
economic losses both in Brazil and in many other areas
of the world. The mould A. alternata is an opportunist
pathogen that is responsible for leaf spot and blight
disease in more than 380 plant species, while fungi of
the genus Colletotrichum cause economically significant
diseases (e.g. anthracnose) in cereals, grasses, fruit and
ornamental plants, mainly in tropical and subtropical
regions (SOUZA JÚNIOR; SALES; MARTINS, 2009). Of
particular interest, however, is the hemibiotrophic fungus
M. perniciosa, the causal agent of witches’ broom disease
in Theobroma cacao L. (GRIFFITH 2004; PURDY;
SCHMIDT, 1996), since this pathogen is responsible for
up to 70% of losses in commercial cocoa plantations in
northern Brazil, and especially in the State of Rondônia.
M. perniciosa mainly infects meristematic vegetative
shoots, inflorescences and fruits resulting in hypertrophied
damaged tissues that are unsuitable for cultivation or
consumption. Although an integrated disease control
management program, including agricultural, chemical,
biologic and molecular strategies, is recommended for M.
perniciosa, a great deal of research in Brazil has focused on
cheap and environmentally friendly methods that could be
suitable for local farmers. Within this context, the applicatio n
of bioactive natural products, in the form of a crude plant
extract or an essential oil, could be more advantageous than
the use of synthetic chemicals since they are likely to be
cheaper to produce, less harmful to the environment and less
toxic to humans (RAHMAN; HOSSAIN; KANG, 2010;
SILVA; BASTOS 2007; STANGARLIN et al., 1999).
Many of the 100 or so species of the genus Ocimum
(Lamiaceae) represent valuable sources of essential oils
that have been used not only in folk medicine, but also
as insect repellents, food flavors and aromatizing agents
in fragrances and perfumes. Paula, Gomes-Carneiro,
Paumgartten (2003), for example, reported that the oil from
Ocimum selloi Benth. was effective against mosquitoes,
showed low acute toxicity, posed no mutagenic risk and did
not cause irritation to human skin. The main compounds
present in the essential oil of O. selloi are methyl chavicol
(estragole; 1-allyl-4-methoxybenzene), trans-anethole,
cis-anethole and caryophyllene (FRANCA et al., 2008;
MORAES et al., 2002; VIEIRA; SIMON 2000).
Considering the small number and high cost
of agrochemicals available for the eradication of
phytopathogens, the emerging signs of resistance
towards traditional fungicides, and the need to reduce
the environmental impact of synthetic agrochemicals,
our research group undertook the task of investigating
the in vitro effects of the essential oil from O. selloi on
the growth of mycelia and the germination of spores of
A. alternata,Colletotrichum gloeosporioides (Penz.)
Penz. & Sacc. and M. perniciosa.
MATERIALS AND METHODS
Plant material
The leaves and flowers of O. selloi were collected
from the Lavras county of the Republic of Brazil. The
plant was identified on the basis of morphological
features and a voucher specimens (number 7474) were
deposited in the herbarium of the Department of Biology
at Universidade Federal de Lavras (UFLA).
Extraction of the essential oil
The fresh leaves and inflorescences portions (100 g) of
O. selloi were subjected to hydrodistillation for 90 min using
a Clevenger type apparatus. The essential oil was dried
over anhydrous magnesium sulphate and preserved in
a sealed vial at 4ºC for further analysis.
Gas chromatography-mass spectrometry (GC-MS)
analysis
The GC-MS analysis of the essential oil was
performed using a SHIMADZU GC-MS (GC-17A and
MS- QP5050A with quadrupole detector) equipped with
DB-5 MS fused silica capillary column (300 × 0.25 mm
i.d.; 0.25 µm film thickness). For GC-MS detection,
an electron ionization system with ionization energy
of 70 eV was used. The carrier gas helium was used at
a constant flow rate of 1.2 ml min-1. Injector and MS transfer
line temperature were set at 250 ºC and 280 ºC, respectively.
The oven temperature was programmed isothermal at 50 ºC
for 2 min, increased at 4 ºC min-1 to 200 ºC, then raised
to 300 ºC at 10 ºC min-1 and finally held isothermal
for 10 min. Diluted samples (20%, v/v, in dichloromethane)
of 0.5 L was injected in the split mode ratio 1:100.
Rev. Ciênc. Agron., v. 46, n. 2, p. 428-435, abr-jun, 2015430
In vitro antifungal activity of Ocimum selloi essential oil and methylchavicol against phytopathogenic fungi
The relative percentage of the oil constituents was
expressed as percentage by peak area normalization.
Identification procedure
The linear retention indices (RI) for all of the
compounds were determined by co-injection of an oil
sample with a solution containing the homologous
series of C8–C32 n-alkanes (Sigma, St Louis, MO, USA)
and application of the equation of Van den Dool and
Kratz (1963). Individual identification of constituents
were based by: (a) comparing RI values and key mass
spectral features with those published in the literature;
(b) comparing individual mass spectra with spectral data
from the NIST/EPA/NHI library (National Institute of
Standards and Technology 1998); (c) co-injection of an
oil sample with authentic standards (ADAMS, 2007).
Fungal pathogens
The plant pathogenic fungi were obtained from
the collection of the Department of Phytopathology at
UFLA. Cultures of each fungal species were maintained
on potato-dextrose agar (PDA) slants and stored at 4 ºC.
The fungal species used in the experiment were A.
alternata (CML 184), C. gloeosporioides (CML 459)
and M. perniciosa (CEPLAC 1188; CML ).
Preparation of spore suspension and standard methyl
chavicol
The spore suspension of A. alternata,C.
gloeosporioides and M. perniciosa were obtained from
their respective 7 days old cultures, mixed with sterile
distilled water to obtain a homogenous spore suspension
of 22 x 104 spore mL-1. Essential oil and standard methyl
chavicol ( 98.9% purity by GC; Riedel-de Haën, Seelze,
Germany) were dissolved in 1% propylene glycol (PG)
separately to prepare the stock solutions, which were
further diluted to prepare test samples.
Bioassays
Mycelial growth assay
In order to evaluate the effects on mycelial growth,
essential oil from O. selloi and standard methyl chavicol
were solubilized separately in 1% propylene glycol (PG)
so as to ease its incorporation into the agar medium. The
essential oil and standard methyl chavicol were tested at
125; 250; 500; or 1,000 ppm. Two different sets of control
(0 ppm of essential oil and PG + 0 ppm of essential oil)
were used. The oil and standard were incorporated to the
autoclaved and cooled (40 ºC) potato dextrose agar medium
(PDA). The medium amended with oil or standard was
then poured into sterilized Petri dishes (9 cm diameter).
A mycelial disc of 7 mm diameter of the test pathogens
taken from a 7 days old culture, using a sterilized cork
borer was placed at the centre of the Petri dish containing
the medium. The plates were then sealed with parafilmTM
and incubated at chamber room at 25 ± 1 ºC under a 12 h
photoperiod. The diameter of the growing mycelia was
performed every two days, time by which the growth of
control would have reached 2/3 of the Petri dish. The
plates were used in triplicates for each treatment.
The growth inhibition of oil and standard trails
compared to control was calculated by mycelial growth
index (MGI), using the following formula: MGI = (D1/ N1) +
(D2/ N2) + ... + (Dn/ Nn), where D1, D2 and Dn represent the
diameters of the mycelia at the first, second and last
measurements, and N1, N2 and Nn represent the number
of days after inoculation.
Additionally, the percentage inhibition of
mycelial growth (IMG) was estimated using the
equation: IMG = [(Dc – Dt) x 100] / Dc, in which Dc and
Dt represent the diameters of the mycelia in the control
and in the treatment, respectively. Bioassays were
repeated six times for each of the oil concentrations.
The minimum concentration at which no visible
growth was observed was defined as the MIC, which was
expressed in ppm and the lethal dose (LD50) defined as
the concentration that is able to inhibit 50% of mycelial
growth, which was expressed in ppm were determined.
Spore germination assay
The second bioassay was designed to evaluate
the effect of different concentrations of essential oil
on the spore germination of the fungal isolates. Glass
depression slides (containing three wells each) were
placed inside Petri dishes that had been lined with wet
filter paper. Each well received an aliquot (100 µL) of
the essential oil solubilized in 1% propylene glycol to
give a final concentration of 125; 250; 500; or 1,000 ppm.
Control without essential oil was tested in the same way.
Subsequently, a 30 µL aliquot of a suspension of spores of
A. alternata (22 x 104 spores.mL-1), C. gloeosporioides
(20 x 104 spores.mL-1) or M. perniciosa (24 x104
spores.mL-1) was added to each well and the glass
slides were incubated at 25 ± 1 ºC for 24 h. At the
end of the incubation period, each well received few
drops of lacto-phenol-cotton blue stain and the slides
were observed under the light microscope. All spores
exhibiting germ tubes, independent of their length, were
considered to be germinating spores. Each treatment
involved three repetitions, each one comprising three
wells, and 200 spores were counted per well to give a
total of 600 spores counted per treatment. The results
were expressed as percentage of spore germination.
Statistical analysis
All experiments were conducted according to a
completely randomized design. Mean values were compared
using Tukey’s test at  0.05 and regression analyses.
Rev. Ciênc. Agron., v. 46, n. 2, p. 428-435, abr-jun, 2015 431
L. C. B. Costa et al.
RESULTS AND DISCUSSION
GC/MS analysis of the essential oil from the leaves
and inflorescences of O. selloi led to the identification of
six main components, namely, two phenylpropanoids and
four sesquiterpenes, representing 93.86% and 6.03% of
the total oil respectively. The identified compounds are
listed in Table 1 according to their elution order on a DB-5
MS capillary column. The major constituent was methyl
chavicol, and this component accounted for more than 93%
of the total oil. The others compounds were methyl eugenol
(0.62%), -caryophyllene (2.22%), germacrene-D (1.33%),
bicyclogermacrene (1.21%) and spathulenol (1.27%).
Table 1 - The principal chemical constituents of the essential oil from leaves and inflorescences of Ocimum selloi
aRetention indices relative to n-alkanes on DB-5 MS capillary column
Figure 1 - Effects of different concentrations of essential oil fromOcimum selloi on the mycelial growth index (MGI) of Alternaria alternata
(y = -0.000001x2 – 0.002150x + 3.885923; R2 = 96.18), Colletotrichum gloeosporioides (y = -0.000001x2 - 0.001445x + 4.437385;
R2 = 99.48) and Moniliophthora perniciosa (y = -0.000002x2 – 0.005871x + 3.496410; R2 = 70.43)
Compound RIaComposition (%)
Methyl chavicol 1198 93.24
Methyl eugenol 1397 0.62
-Caryophyllene 1418 2.22
Germacrene-D 1480 1.33
Bicyclogermacrene 1494 1.21
Spathulenol 1575 1.27
Phenylpropanoids 93.86
Sesquiterpenes 6.03
TOTAL 99.89
The essential oil from O. selloi was able to inhibit
mycelial growth in all three fungal species assayed. The
toxic effect was in a dosage response manner, i.e. the
higher the concentration of essential oil the greater was
the reduction in the growth of mycelia (Fig. 1 and Fig.2).
For A. alternata and C. gloeosporioides, maximum IMG
values of 45.7 and 13.2%, respectively, were attained when
the concentration of essential oil was 1,000 ppm (Table 2).
Of more interest, however, was the effect of the essential
oil on the mycelial growth of M. perniciosa, which was
inhibited by 100% at oil concentrations of 500 and 1,000
ppm (Fig. 2c), and the relative low MIC and LD50 values
obtained (Table 2). As shown in Fig. 3, pure methyl
Rev. Ciênc. Agron., v. 46, n. 2, p. 428-435, abr-jun, 2015432
In vitro antifungal activity of Ocimum selloi essential oil and methylchavicol against phytopathogenic fungi
chavicol or estragole also reduced the growth of mycelia
of M. perniciosa, and complete inhibition of growth was
observed at 1,000 ppm.
Figure 2 - Effects of different concentrations of essential oil from
Ocimum selloi on the growth of mycelia of Alternaria alternata
(a), Colletotrichum gloesporioides (b) and Moniliophthora
perniciosa (c). Each panel shows - (1) 0 ppm of essential oil
(control), (2) PG + 0 ppm of essential oil (control), (3) 125 ppm,
(4) 250 ppm, (5) 500 ppm, and (6) 1000 ppm. Bar = 5 cm
Figure 3 - Effects of different concentrations of methyl
chavicol (standard) on the mycelial growth index (MGI) -
(y = -5E- 06X2 + 0.0027x + 2.5862; R2 = 99.54**) a) and
mycelia growth to Moniliophthora perniciosa (b) – Each panel
shows - (1) 0 ppm of methyl chavicol (control), (2) PG + 0 ppm of
methyl chavicol (control), (3) 125 ppm, (4) 250 ppm, (5) 500 ppm,
and (6) 1000 ppm of methyl chavicol. Bar = 5 cm
The essential oil from O. selloi affected the
germination of spores of C. gloeosporioides and M.
perniciosa at all concentrations tested, although the largest
reductions in germination (93 and 87%, respectively) were
observed at 1,000 ppm (Figure 4). In complete contrast,
the germination of A. alternata spores was not affected by
any of the treatments applied.
The essential oils are promising natural anti-
fungal agents with potential applications in agriculture to
control phytopathogenic fungi causing severe destruction
of crops. The numerous aromatic and medicinal plants
are considered to be non-phytotoxic compounds and
potentially effective against plant pathogenic fungi
(CAROVI-STANKO et al., 2010).
The hydrodistillation of the leaves and flowers
part of O. selloi gave light yellowish oil with the major
components of the oil having phenylpropanoids and
sesquiterpenes. In recent years, several researchers
have reported that mono - and sesquiterpenes have
Rev. Ciênc. Agron., v. 46, n. 2, p. 428-435, abr-jun, 2015 433
L. C. B. Costa et al.
Table 2 - Percentage inhibition of mycelial growth (IMG) of different concentrations of the essential oil from Ocimum selloi tested
against three fungal isolates, minimum inhibitory concentration (MIC) and lethal dose (LD50)
Fungal strains
IMG (%)
MIC (ppm) LD50 (ppm)Essential oil concentration (ppm)
125 250 500 1,000
Alternaria alternata 0.6 1.7 19.2 45.7 > 1000 585.17
Colletotrichum gloeosporioides 0.0 4.0 3.9 13.2 > 1000 752.23
Moniliophthora perniciosa 5.4 3.2 100.0 100.0 250 – 500 415.25
Figure 4 - Effects of different concentrations of essential oil from Ocimum selloi on the spore germination from Alternaria alternata
(y = 0.000003x2 – 0.005192x + 100.034615; R2 = 0.87), Colletotrichum gloeosporioides (y = 0.000073x2 – 0.166687x + 100.226923;
R2 = 0.99) and Moniliophthora perniciosa (y = 0.000087x2 – 0.151606x + 75.846154; R2 = 0.99)
enormous potential to inhibit microbial pathogens
(CAROVI- STANKO et al., 2010; RAHMAN; HOSSAIN;
KANG, 2010). Also, was reported a significant difference
in antifungal activity among functional groups, that
phenylpropanoids compounds were more toxic than
hydrocarbons (KIM et al., 2008).
The results show that the essential oil of O.
selloi was able to inhibit the growth of mycelia of
the three fungal isolates studied, with the order of
sensitivity to the oil being M. perniciosa bigger than
C. gloeosporioides and A. alternata. Furthermore, the
essential oil caused a reduction in the germination of
spores of M. perniciosa at all concentrations tested,
thus indicating a possible application of this natural
product in the treatment of infected cacao crops.
Since the major constituent (> 93%) of the essential
oil was methyl chavicol, the fungicidal activity of this
phenylpropanoid against M. perniciosa was also evaluated.
The results revealed that the essential oil and pure methyl
chavicol were similarly effective in causing complete
inhibition of mycelial growth at a concentration of 1,000
ppm. Previous studies demonstrated that the essential oil from
another species of Ocimum, namely O. basilicum L.,as well
as the pure phenylpropanoids methyl chavicol and eugenol,
were able to reduce significantly the mycelial growth of the
phytopathogenic fungus Botrytis fabae Sardiña (OXENAHM;
SVOBODAL; WALTERS, 2005). Additionally, a number
of other phenylpropanoids have been reported to exhibit
antifungal activity, including apiole (MEEPAGALA
et al., 2005; RAZZAGHI-ABYANEH et al., 2007) and the
Rev. Ciênc. Agron., v. 46, n. 2, p. 428-435, abr-jun, 2015434
In vitro antifungal activity of Ocimum selloi essential oil and methylchavicol against phytopathogenic fungi
related nothoapiole (LAOUER et al., 2009). In this study, the
essential oil showed a remarkable anti-fungal activity against
M. perniciosa, which could be attributed to the presence of
phenylpropanoids mainly methyl chavicol. This was further
supported by our findings, when was used standard methyl
chavicol at 1,000 ppm. Although, essential oil at 500 ppm
had efficient inhibition effect against M. perniciosa, but the
standard at 500 ppm had not effect. It is possible that the minor
components might be involved in some type of synergism
with the other active compounds. Volatile compounds, such
as -caryophyllene, spathulenol (RAHMAN; HOSSAIN;
KANG, 2010), methyl eugenol (KIM et al., 2008) have been
claimed to contain the strong antifungal properties.
Various studies have shown that the essential oils
from many aromatic or medicinal plants are active against
a range of phytopathogenic fungi. Thus oils from Pimenta
racemosa (Mill.) J.W.Moore and Thymus vulgaris L. were
able to inhibit completely the growth of the phytopathogenic
fungi Phytophthora cactorum (Lebert & Cohn) J. Schröt.
and Cryphonectria parasitica (Murrill) M.E. Barr
(KIM et al., 2008), whilst the essential oil from Aloysia
gratissima (Gillies & Hook.) Tronc. was active against C.
gloeosporioides and Fusarium oxysporum Schltdl. in a dose-
dependent fashion (CARDOSO, 2005). Treatment of Botrytis
cinerea Pers., F. oxysporum and Bipolaris sorokiniana (Sacc.)
Shoemaker with essential oil derived from Eucalyptus spp. at
a concentration of 500 ppm resulted in a significant reduction
of mycelia growth (SALGADO et al., 2003).
With regard to M. perniciosa, inhibition of mycelia
growth and spore germination by the essential oils and leaf
extracts from a number of species of Piper has been described
(SILVA; BASTOS, 2007). Thus, the essential oil from P.
aduncum L. completely inhibited mycelia growth and spore
germination in the phytopathogenic fungus when applied
at 50 and 100 ppm, respectively, whilst oils from P. dilatatum
Rich., P. callosum Opiz and P. marginatum var. anisatum
C.DC. inhibited M. perniciosa spore germination at
concentrations of 400, 400 and 500 ppm, respectively
(SILVA; BASTOS, 2007). Additionally, the essential oils
P. callosum, P. marginatum var. anisatum and P. enckea
C.DC. exhibited antifungal activities against M. perniciosa,
Phytophthora palmivora (E.J. Butler) E.J. Butler and P.
capsici Leonian, all of which attack cacao plants (SILVA;
BASTOS, 2007). The present study, however, reports for the
first time the antifungal activity of an individual component
of an essential oil, namely, pure methyl chavicol against M.
perniciosa. Although the present findings suggest a possible
application of this natural product in the treatment of infected
cacao crops, further in vivo studies are required in order to
confirm the efficacy of this natural fungicide and to determine
its safety. Kim et al. (2008) reported there was a significant
difference in antifungal activities among phenylpropanoids,
that the position of the double bond of the prophenyl group is
also important for antifungal activity.
CONCLUSIONS
1. The essential oil from O. selloi, when applied at a
concentration of 1,000 ppm, is able to inhibit mycelia growth
(100%) and spore germination (87%) of M. perniciosa;
2. The antifungal activity is attributed to the major
constituent methyl chavicol, which showed activity
at 1,000 ppm similar to that of the essential oil.
3. The essential oil from O. selloi was more effective to
inhibit mycelia growth and spore germination of M.
perniciosa that C. gloesporioides and A. alternata.
ACKNOWLEDGMENTS
The authors wish to thank the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) for
fellowships awarded to two of us (JEBPP and PBA), and the
Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG)
and the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) for financial support for the study.
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... The use of natural products such as essential oils has shown promising results in the treatment of forest seeds species (Pereira et al., 2016;Gomes et al., 2016;Medeiros et al., 2016). Essential oils are aromatic and volatile products of the secondary metabolism of some plants, and usually exhibit antimicrobial properties (Bajpai et al., 2011;Costa et al., 2015). The questions we asked in conducting this study are: Can Paineira seeds without any treatment germinate? ...
... Similar results with Lemongrass oil were found by Souza Junior et al. (2009) with a concentration of 1 μL mL -1 totally inhibited the mycelial growth of the fungus C. gloeosporioides. Work with alternative control using oils and plant extracts has brought satisfactory results in the control of seed pathogenic fungi (Costa et al., 2015;Gomes et al., 2016;Medeiros et al., 2016). The essential oil is one of the most promising groups of natural compounds for the development of safer antifungal agents (Manssouri et al., 2016;Starović et al., 2017). ...
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... Essential oils extracted from plants are generally assumed to be more acceptable and less hazardous than synthetic compounds as they possess bioactive properties, due to the presence of many chemical components such as eugenol, camphor, camphene, caryophyllene, phellandrene, etc. (Bakkali et al., 2008;Zaker, 2016). In several previous studies, it has been recorded that essential oils of cinnamon (Cinnamomum zeylanicum) leaf and bark, clove (Syzygium aromaticum), and basil (Ocimum basilicum) as very eff ective sources of antifungal compounds (Ranasinghe, 2003;Sukatta et al., 2008;Abeywickrama et al., 2009;Costa et al., 2015). ...
... All these chemical constituents have previously been reported as bioactive antifungal compounds against many postharvest pathogenic fungi. Methyl chavicol, which is the most abundant component present in basil oil was found to be antifungal against two postharvest pathogenic fungi viz, C. gloeosporioides and Alternaria alternata (Costa et al., 2015). Eugenol, which was recognised as one of the most eff ective bioactive components present in the test oils has previously been reported for its strong antifungal activity against C. musae, F. proliferatum, and Botrytis cinerea (Herath & Abeywickrama, 2008;Wang et al., 2010). ...
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Karutha Colomban is one of the most delightful mango varieties popular among Sri Lankan consumers. A significant postharvest loss of mango takes place every season due to diseases including stem-end rot (SER), which is caused by a group of endophytic fungal pathogens. In this research, in vitro antifungal efficacy of different concentrations of essential oils of basil, clove, and cinnamon were evaluated for their ability to control SER causing fungal pathogens of mango (cv. Karutha Colomban) as bio-safe alternatives to conventional fungicides, by conducting liquid and disc volatilisation bioassays. Major bioactive compounds of the selected essential oils were identified by gas chromatography-mass spectroscopy (GC-MS). Basil and cinnamon bark oils (0.20 – 0.30 μL/mL) in liquid bioassay showed high effi cacy against Lasiodiplodia theobromae, while basil and cinnamon leaf oils (0.40 – 0.60 μL/mL) successfully inhibited Pestalotiopsis sp. Cinnamon bark oil (0.60 μL/mL) was identifi ed as the most effective oil against Phomopsis sp. According to disc volatilisation bioassay, vapour of cinnamon oils (0.20 – 0.40 μL/mL) was the most effective in controlling L. theobromae. Pestalotiopsis sp. was efficiently controlled by clove and cinnamon bark oil (0.20 – 0.60 μL/mL) vapour. In vapour phase, clove and cinnamon oils (0.40 μL/mL) were the most effective against Phomopsis sp. According to GC-MS characterisation, methyl chavicol was the most abundant antifungal component in basil oil while it was (E)-cinnamaldehyde in cinnamon bark oil. Moreover, eugenol displayed the highest abundance in clove and cinnamon leaf oils. Based on in vitro studies, it could be concluded that cinnamon bark oil in liquid and vapour phases demonstrated a higher antifungal efficacy among the tested essential oils in controlling fungal pathogens causing SER of mango.
... where dC and dT are the mean diameters of the mycelial growth in the control and treated plates, respectively (Costa et al., 2015). ...
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The green silver nanoparticles (green AgNPs) exhibit an exceptional antimicrobial property against different microbes, including bacteria and fungi. The current study aimed to compare the antifungal activities of both the crude aqueous extract of Portulaca oleracea or different preparations of green AgNPs biosynthesized by mixing that aqueous extract with silver nitrate (AgNO3). Two preparations of the green AgNPs were synthesized either by mixing the aqueous extract of P. oleracea with silver nitrate (AgNO3) (normal AgNPs) or either irradiation of the AgNPs, previously prepared, under ⁶⁰Co γ-ray using chitosan (gamma-irradiated AgNPs). Characterization of different AgNPs were tested by Zeta potential analyzer, Ultraviolet (UV) Visible Spectroscopy, and Fourier-Transform Infrared (FTIR) spectrometry. Three different plant pathogenic fungi were tested, Curvularia spicifera, Macrophomina phaseolina, and Bipolaris sp. The antifungal activities were evaluated by Transmission Electron Microscope (TEM) for either the crude aqueous extract of P. oleracea at three doses (25%, 50%, and 100%) or the newly biosynthesized AgNPs, normal or gamma-irradiated. With a few exceptions, the comparative analysis revealed that the irradiated green AgNPs at all three concentrations showed a relatively stronger antifungal effect than the normal AgNPs against all the three selected fungal strains. UV-visible spectroscopy of both preparations showed surface plasmon resonance at 421 nm. TEM results showed that both AgNPs were aggregated and characterized by a unique spherical shape, however, the gamma-irradiated AgNPs were smaller than the non-irradiated AgNPs (0.007 to 0.026 µM vs. 0.009 to 0.086 µM). TEM photographs of the fungal strains treated with the two AgNPs preparations showed flaccid structures, condensed hyphae, and shrunken surface compared with control cells. The data suggested that the biosynthesized P. oleracea AgNPs have antifungal properties against C. spicifera, M. phaseolina, and Bipolaris sp. These AgNPs may be considered a fungicide to protect different plants against phytopathogenic fungi.
... The strong antifungal activity of basil oil may be due to its main compositions, namely, methyl chavicol (61.3%) and linalool (24.8%) ( Table 1). Methyl chavicol was proven to be a strong antifungal agent against Moniliophthora perniciosa with minimum fungicidal concentration (MFC) of 1000 ppm [73] and Uromyces viciae-fabae with MFC of 1000 ppm [19]. Similarly, linalool showed the strong inhibitory activity against Uromyces viciae-fabae with MFC of 1000 ppm [19]. ...
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Anthracnose disease caused by Colletotrichum spp. makes heavy losses for post-harvest mangoes of Cat Hoa Loc variety during storage, packaging, and transportation. The synthetic fungicides are commonly used to control the disease, but they are not safe for consumers’ health and environment. This study was aimed to investigate the use of essential oils (EOs) as the safe alternative control. Pathogen was isolated from the infected Cat Hoa Loc mangoes and identified by morphology and DNA sequencing of the ITS region. Six EOs (cinnamon, basil, lemongrass, peppermint, coriander, and orange) were chemically analyzed by GC–MS. The antifungal activity of EOs was studied in vitro and in vivo. The results showed that the isolated pathogen was Colletotrichum acutatum. Cinnamon, basil, and lemongrass EOs effectively inhibited the growth of C. acutatum in descending order of cinnamon, basil, and lemongrass. However, they (except basil oil) severely damaged fruit peels. The antifungal activity was closely related to the main compounds of EOs. Basil EOs effectively controlled anthracnose development on Cat Hoa Loc mangoes artificially infected with C. acutatum, and its effectiveness was comparable to that of fungicide treatment. Consequently, basil EOs can be used as a biocide to control anthracnose on post-harvest Cat Hoa Loc mangoes.
... where OD sample-optical density at 540 nm as the mean value of triplicate readings for EOs in the presence of the selected bacteria; OD negative control-optical density at 540 nm as the mean value of triplicate readings for the selected bacteria in BHI. BGR/MGR indicators are calculated values expressed as percentages of growth or inhibition, indicators used previously in several other research articles [56,[60][61][62]. ...
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The purpose of this study was to analyze the chemical composition and antimicrobial activity of some thymus populations collected from five different locations in Western Romania. The chemical compositions of the essential oils (EOs) were studied through GC–MS, and the biological activities were evaluated using the microdilution method. The EO yield ranged between 0.44% and 0.81%. Overall, 60 chemical compounds were identified belonging to three chemotypes: thymol (three populations), geraniol (one population) and carvacrol (one population). Thymus vulgaris L. is distinguished by a high content of thymol, while species of spontaneous flora (Th. odoratissimus and Th. pulegioides) contain, in addition to thymol, appreciable amounts of carvacrol and geraniol. The antimicrobial activity of each the five oils was tested on Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes (ATCC 19615), Esherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Shigella flexneri (ATCC 12022), Salmonella typhimurium (ATCC 14028), Haemophilus influenzae type B (ATCC 10211), Candida albicans (ATCC 10231) and Candida parapsilopsis (ATCC 22019). The EOs showed biological activity on Gram-positive/Gram-negative/fungal pathogens, the most sensitive strains proving to be S. pyogenes, S. flexneri, S. typhimurium and C. parapsilopsis with an MIC starting at 2 µL EO/100 µL. The species sensitive to the action of Thymus sp. from culture or spontaneous flora are generally the same, but it should be noted that T. odoratissimus has a positive inhibition rate higher than other investigated EOs, regardless of the administered oil concentration. To date, there is no research work presenting the chemical and antimicrobial profiling of T. odoratissimus and the correlations between the antimicrobial potential and chemical composition of wild and cultivated populations of thyme (Thymus sp.) growing in Western Romania.
... The phytochemical studies of Piper, Pimenta, Plectranthus and Psidium have described the presence of monoterpenic and sesquiterpenic hydrocarbons, oxygenated monoterpenes and sesquiterpenes, phenylpropanoids, esters, fatty acids, alcohols, aldehydes, phenolic acids and flavonoids that represent a rich source of biocides (Chaudhari et al., 2018;Estrada-Reyes et al., 2018;Weli et al., 2018). In particular, several of these compounds have been reported to have antifungal activity on C. gloeosporioides, C. acutatum, and other phytopathogens (Alzate et al., 2009;Numpaque et al., 2011;Costa et al., 2015;Hong et al., 2015). To our knowledge, the antifungal activity of terpenic and phenolic extracts obtained from wild aromatic species against postharvest fungal pathogens has been rarely explored. ...
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... The EO of Ocimum sp., whose yield was 0.30% v/w, presented methyl eugenol (77.8 wt.%) as the major compound, followed by elemicin (5.6 wt.%), and α-farnesene (4.5 wt.%). Costa et al. (45) reported, for individuals of O. selloi collected in Southeastern Brazil, methyl chavicol (estragole) as the major compound (93.2 wt.%), methyl eugenol presented itself as a minor compound (0.6 wt.%). However, the only difference between these two compounds is the insertion of a second methoxy group (H 3 C-O-) in the ortho position of the aromatic ring. ...
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This work aimed to evaluate the chemical composition, yield, and antifungal activity of the essential oils (EO) of native species from the ‘Campos de Cima da Serra’ region, South Brazil. The species Aloysia gratissima, Aloysia lycioides, Blepharocalyx salicifolius, Cunila angustifolia, Myrciaria delicatula, Myrcia brasiliensis, Ocimum sp., and Schinus polygama had an EO yield higher than 0.1% v/m. The in vitro antifungal activity of these oils was tested on the fungi Alternaria alternate, Fusarium oxysporum, Botrytis cinerea, Sclerotinia sclerotiorum, and Colletotrichum gloeosporioides, with an EO concentration of 0.1% v/v. Chemically, the EO of Ocimum sp. presented the highest dissimilarity compared to other species; the EOs of M. delicatula and M. brasiliensis presented the highest degree of similarity. Ocimum sp. EO inhibited the growth of all fungi; the EO of B. salicifolius had no inhibitory effect. The remaining oils had an intermediate antifungal activity which also depended on the fungi species.
... For A. alternata and C. gloeosporioides, the percentage of maximal mycelial inhibition at a concentration of 1000 ppm was 45.7 and 13.2%, respectively. At concentrations of 1000 ppm and 500 ppm, the essential oil inhibited 100% growth of M. perniciosa (Costa et al., 2015). ...
... Essential oils are highly complex mixtures of volatile aromatic compounds synthesized by plants, which have diverse ecological functions, including the attraction of pollinators and defense mechanisms against harmful microorganisms (Bakkali et al., 2008;Medeiros et al., 2011;Li et al., 2013;Amaral et al., 2015;Costa et al., 2015;De Almeida et al., 2018). Monoterpenes, aldehydes, allyl phenols, alcohols, acids, and esters are the main components of plant EOs (Prakash et al., 2015;De Almeida et al., 2018). ...
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This study assessed the feasibility of using essential oils (EOs) against Colletotrichum nymphaeae inciting strawberry anthracnose. Two EOs, extracted from Allium sativum (garlic) and Rosmarinus officinalis (rosemary), were selected because their fungicide efficacy was already well characterized under laboratory and greenhouse conditions. We characterized both EOs in terms of efficacy and impact on qualitative traits and sensory quality of strawberry fruit. The gas chromatography-mass spectrometry analysis confirmed the Diallyl trisulfide (29.08%) and (α)-pinene (15.779%) as the main components of A. sativum and R. officinalis EOs, respectively. Both A. sativum and R. officinalis EOs significantly inhibited the mycelial growth and conidial germination of C. nymphaeae in contact and vapor assays compared with untreated control. However, EC 50 assay indicated A. sativum EO was more effective than R. officinalis EO against the pathogen. Malformations of the vegetative structures of the pathogen exposed to both EOs were revealed as shriveled, collapsed, and swelling mycelia in the cultures. Both EOs confirmed their efficacy under in vivo and greenhouse conditions; in fact, they significantly reduced the development of fruit decay and anthracnose disease incidence and severity, compared with untreated controls. Both EOs preserved sensory attributes and quality parameters of strawberry fruit including firmness, total soluble solids, ascorbic acid, antioxidant activity, and anthocyanin, but may leave unwanted smells. These findings suggest that two EOs can potentially represent an alternative to synthetic chemical fungicides against C. nymphaeae preserving fruit quality factors, although their cost and the impact on the fruit odor must be carefully taken into consideration before developing a commercial product.
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Ocimum species represent commercially important medicinal and aromatic plants. The essential oil biosynthesized by Ocimum species is enriched with specialized metabolites specifically, terpenoids and phenylpropanoids. Interestingly, various Ocimum species are known to exhibit diverse chemical profiles, and this chemical diversity has been at the center of many studies to identify commercially important chemotypes. Here, we present various chemotypes from the Ocimum species and emphasize trends, implications, and strategies for the quality and yield improvement of essential oil. Globally, many Ocimum species have been analyzed for their essential oil composition in over 50 countries. Asia represents the highest number of chemotypes, followed by Africa, South America, and Europe. Ocimum basilicum L. has been the most widespread and well-studied species, followed by O. gratissimum L., O. tenuiflorum L., O. canum Sims, O. americanum and O. kilimandscharicum Gürke. Moreover, various molecular reasons, benefits, adverse health effects and mechanisms behind this vast chemodiversity have been discussed. Different strategies of plant breeding, metabolic engineering, transgenic, and tissue-culture, along with anatomical modifications, are surveyed to enhance specific chemotypic profiles and essential oil yield in numerous Ocimum species. Consequently, chemical characterization of the essential oil obtained from Ocimum species has become indispensable for its proper utilization. The present chemodiversity knowledge from Ocimum species will help to exploit various applications in the industrial, agriculture, biopharmaceutical, and food sectors. Supplementary information: The online version contains supplementary material available at 10.1007/s11101-021-09767-z.
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With the use of irrigation and new hybrids of cauliflower, it is possible to get production during all the year with hight yield. However, the crop has been affected by diseases, as the dark rot caused by X. campestris pv. campestris. The objective of this research work was to study the potential of Mikania glomerata for the control of this disease. Alcoholic extract 50 ºGL of M. glomerata was evaluated regarding to: in vitro antimicrobial activity through bacterial growth in 100, 250, 500 and 1000 mg L-1 of the alcoholic extract; induction of local or systemic resistance in 25 days old cauliflower, with spray of alcoholic extract concomitantly and three days before the inoculation with the pathogen (water and bordeau mixture were used as control); peroxidases activity in leaves of cauliflower treated and not treated, and harvested concomitantly, 24, 48 and 72 hours after spraying the alcoholic extract and also after inoculation. The alcoholic extract of M. glomerata showed inhibition of the bacterial growth in vitro at the concentrations of 250, 500 and 1000 mg L-1. The concentrations of 500 mg L-1 and 1000 mg L-1 inhibited 24% and 38% of the bacterial growth. This inhibition could be due to antibacterial compounds in the alcoholic extract. An inhibition of the disease in vivo occurred only in the leaves treated with 100 and 500 mg L-1 of alcoholic extract when applied concomitantly with the bacteria. This result was similar to bordeau mixture, indicating a control by direct antimicrobial activity. There was no systemic resistence induction for all treatments. The peroxidases induction was due to infectious pathogen process and not to the treatments with alcoholic extract. The results indicate the potential of M. glomerata alcoholic extract for the preventive control of cauliflower dark rot disease.Com a prática da irrigação e novos híbridos de couve-flor, é possível produzir durante todo o ano e com alta produtividade. Mas, a cultura tem sido afetada por doenças a exemplo da podridão negra causada por Xanthomonas campestris pv. campestris, fomentando novas pesquisas para seu controle. Com o objetivo de verificar o potencial de Mikania glomerata no controle dessa doença, a tintura etanólica 50 oGL dessa planta medicinal foi avaliada quanto: atividade ntimicrobiana in vitro através do crescimento bacteriano em tubos de ensaio contendo 100, 250, 500 e 1000 mg L-1 da tintura; indução de resistência local ou sistêmica em plantas de couve-flor aos 25 dias de idade, em casa de vegetação, através da pulverização de tintura oncomitantemente e três dias antes da inoculação com o patógeno, sendo água e calda bordaleza tratamentos controle; atividade de peroxidases em folhas tratadas e não tratadas de couve-flor, colhidas concomitantemente e as 24, 48 e 72 h da pulverização da tintura e, após pulverizaçãoinoculação. A tintura etanólica, in vitro, promoveu inibição no crescimento bacteriano, a partir da concentração de 250 mg L-1. Nas concentrações de 500 mg L-1 e 1000 mg L-1 foram observadas, respectivamente, 24% e 38% de inibição do crescimento bacteriano. Nas plantas de couve-flor foi observada redução da doença apenas em folhas tratadas com 100 e 500 mg L-1 de tintura, plicada concomitantemente à inoculação, comportamento este semelhante ao da calda bordaleza, ndicando que o controle através da tintura de guaco é através de atividade antimicrobiana direta. Ficou indicado que a indução de peroxidases ocorreu devido ao processo infeccioso e não em função dos tratamentos com tintura etanólica de guaco. Estes resultados indicam o potencial da tintura de guaco para o controle preventivo da podridão negra em couve-flor.
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