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

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.

Full text

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.

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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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