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Control of Fusarium verticillioides using Palmarosa Essential Oil (Cymbopogon martinii)

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This study aimed to evaluate the fungi toxic effect of palmarosa essential oil (Cymbopogon martinii) on mycelial growth of Fusarium verticillioides in vitro and treatment of corn seeds. For the in vitro experiment the essential oil was added to the culture medium and poured into Petri dishes, using seven different oil concentrations (0.0125, 0.025, 0.05, 0.1 and 0.2%), 0.0% was the negative control, and we used Thiram as a positive control. Discs of culture medium with fungal mycelium were inoculated into the center of the plates and incubated for seven days at 27 ± 2°C. Growth was evaluated and the percentage of mycelial growth inhibition and mycelial growth rate index were calculated. For corn seed experiment, seven different oil concentrations (0.1, 0.2, 1.0, 3.0, 5.0, and 6.0%) were used, and 0.0% was the negative control, and Thiram the positive control. The artificial inoculation was carried out in fungi colonies and the seed sanity test performed. The percentage of seeds infected by the fungus was evaluated after seven days. Under in vitro conditions, palmarosa oil reduced the mycelial growth of F. verticillioides at all concentrations tested. The highest dose, 0.2%, totally inhibited fungus growth. In seed treatment, the oil significantly reduced the percentage of infected seeds above 3.0% of concentration.
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Int.J.Curr.Microbiol.App.Sci (2019) 8(5): 484-494
484
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 8 Number 05 (2019)
Journal homepage: http://www.ijcmas.com
1Agroindustrial Systems, Federal University of Campina Grande, Pombal, PB, Brazil
2Environmental Engineer, Federal University of Campina Grande, Pombal, PB, Brazil
3Department of Agronomy, Rural Federal University of Pernambuco, Recife, PE, Brazil
4Postgraduate in Agronomy/Plant Protection, Rural Federal University of Semiarid,
Mossoró, RN, Brazil
5Departament of Agronomy, Federal University of Campina Grande, Pombal, PB, Brazil
6Phytopathology Laboratory, Federal University of Campina Grande, Pombal, PB, Brazil
*Corresponding author
Control of Fusarium verticillioides using Palmarosa
essential oil (Cymbopogon martinii)
Kevison Romulo da Silva Franca1*, Alda Leaby dos Santos Xavier1, Flavia Mota de
Figueiredo Alves1, Tiago Silva Lima1, Ionaly Gomes de Araújo1, Lídia Pinheiro da
Nóbrega1, Antônio Hugo Costa Nascimento2, Antônio Francisco de Mendonça Júnior3,
Ana Paula Medeiros dos Santos Rodrigues4, Fernandes Antônio de Almeida5
and Tiago Augusto Lima Cardoso6
https://doi.org/10.20546/ijcmas.2019.805.057
Original Research Article
A B S T R A C T
Introduction
Corn (Zea mays L.) is the second most
important crop of Brazilian agribusiness. It is
estimated the production of 96 million tons in
the harvest of 2018/2019, characterizing the
country as the third largest producer and
second in the export classification of this
K e y w o r d s
Alternative control,
Mycelial growth,
Plant diseases,
Seeds pathology,
Zea mays L.
Article Info
Accepted:
07 April 2019
Available Online:
10 May 2019
Int.J.Curr.Microbiol.App.Sci (2019) 8(5): 484-494
485
cereal (Conab, 2019). Almost all production is
consumed internally (Alves, 2007), allocating
around 70 to 80% to animal feed and feed
industry: 51% are directed to the poultry
sector, 33% to swine, 11% to livestock, and
5% for food supplementation of other animals
(Queiroz et al., 2012).
Due to the economic importance of maize and
the adoption of technologies by producers, the
demand for high-quality seeds has increased,
causing companies to adopt quality standards
that are more stringent than those established
by the certification system (Fantazzini et al.,
2016). Despite all the technology employed,
maize is a crop susceptible to several diseases
that reduce productivity and lead to
significant economic losses.
In Brazil, fungi from the genus Fusarium
cause the main diseases associated with
maize, mainly the species Fusarium
verticillioides and F. graminearum, which
cause root rot, seedling death, stem rot, and
stem rot, responsible for losses in infected
crops (Munkvold, 2003). F. verticillioides has
been found in corn seeds produced in the
country, so infected seeds represent survival
sites and important vehicle of dissemination
of phytopathogen (Ribeiro et al., 2005;
Nerbass et al., 2008).
The chemical treatment of seeds with
fungicides is the main measure adopted
(Goulart and Melo Filho, 2000). However,
this conventional practice has caused serious
environmental, economic and public health
problems, since residues remain for a long
time in the environment, contaminating the
natural resources and crops produced, which
reach consumers with noxious substances
(Cruz and Farias, 2017).
The use of alternative products with similar
effects to conventionally used chemical
pesticides, but not harmful to the environment
or to human health is a present need. Among
the products widely tested, the essential oils
extracted from aromatic plants have shown
satisfactory antifungal effect in the in vitro
control of phytopathogens (Sousa, Serra and
Melo, 2012; França et al., 2018; Ugulino et
al., 2018; Nóbrega et al., 2019), and seed
treatment (Hillen et al., 2012), with low
toxicity, rapid degradation by the
environment, and safer to human health (Silva
et al., 2018). The essential oils can be used in
an integrated way to other management
techniques contributing to the reduction of the
use of synthetic chemical inputs (Machado,
Silva and Oliveira, 2007).
The essential oil of palmarosa (Cymbopogon
martinii) has an antifungal activity well
documented in the literature, and its
biological activity has been studied in the last
years. The main constituents of palmarosa oil
are geraniol (82%), geranyl acetate (9%),
linalool (2%), tran-β-ocimene (1%), and
geraniol is the main constituent associated
with its antimicrobial activity (Scherer et al.,
2009). The use of palmarosa oil present
promising results in the control of
phytopathogens, such as Fusarium solani
(Nascimento, Vieira and Kronka, 2016),
Phomopsis azadirachtae (Prasad et al., 2012)
and Rhizoctonia solani (Hillen et al., 2012)
Taking in account the importance of seeds to
the production system and the susceptibility to
phytopathogens (Berger, Sinha and Roitsch,
2007), phytosanitary treatments with
environmentally safe products are current
market demand, avoiding negative influence
by chemical fungicides (Hillen et al., 2012).
Therefore, in this study, we aimed to evaluate
the fungi toxic potential of palmarosa
essential oil in the inhibition of the mycelial
growth of Fusarium verticillioides as well as
its efficiency in the maintenance of the
sanitary quality of corn seeds.
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486
Materials and Methods
Experiment location and
materials
The work was conducted at the Center of
Science and Technology Agrifood (CCTA) of
the Federal University of Campina Grande
(UFCG), Campus of Pombal. The
experiments were carried out in the
Phytopathology laboratory, from January to
March, 2019.
We used the strain 3434 of Fusarium
verticillioides yielded by the collection of
phytopathogenic fungi Prof. Maria Menezes
of the Federal Rural University of
Pernambuco, preserved until the assay in
sterile distilled water by the Castellani
method.
The essential oil of palmarosa (Cymbopogon
martinii) was used, obtained by the steam
distillation process, according to the
techniques adopted by FERQUIMA -
Indústria e Comércio Limitada, Vargem
Grande / São Paulo. Hybrid corn seeds AG
1051 were purchased at a commercial house
in the city of Pombal, with a minimum purity
of 98% and a minimum germination of 85%.
Experimental design
Effect of palmarosa essential oil
(Cymbopogon martinii) on Fusarium
verticillioides in vitro
The experiment had a completely randomized
design consisting of seven treatments (5 oil
concentrations, 1 negative control and 1
positive control) in five replicates each. The
treatments consisted of autoclaved medium
supplemented with pure palmarosa essential
oil at different concentrations (0.0125, 0.025,
0.05 and 0.1 and 0.2%), the negative control
(0.0%), and the positive control consisting of
commercial fungicide supplementation
Thiram at the manufacturer's recommended
concentration (1 mL L-1).
The treatments were incorporated into the
autoclaved flux-BDA (Potato Dextrose Agar)
culture medium. After cooling, the medium
was poured into 7.5 cm diameter Petri dishes
under aseptic conditions. Disks of 1 cm
diameter culture medium containing mycelia
of the fungus were transferred to the center of
each plate containing the treatments. The
plates were then wrapped in plastic film and
incubated in a B.O.D type oven (Biochemical
Oxygen Demand) at a temperature of 27 ± 2 °
C.
Colony growth was measured daily until the
colony took the entire surface of the culture
medium from one of the plaques or in a
maximum period of 7 days. Mycelial growth
evaluation consisted of daily measurements of
the diameter of the colonies obtained through
the average of two perpendicular
measurements, using a graduated ruler,
resulting in the average daily growth for each
repetition of each treatment.
The percentage of mycelial growth inhibition
(PGI; Bastos, 1997) and mycelial growth rate
index (IMGS; Oliveira, 1991) were calculated
according to formulas (1) and (2):
(1)
(2)
Effect of palmarosa essential oil
(Cymbopogon martinii) on Fusarium
verticillioides in maize seeds
The experimental consisted of a completely
randomized design with 8 treatments of
sterilized distilled water solutions
supplemented with palmarosa essential oil at
the concentrations 0.1, 0.2, 1.0, 3.0, 5.0 and
Int.J.Curr.Microbiol.App.Sci (2019) 8(5): 484-494
487
6.0%, a negative control, 0.0%, and a positive
control supplemented with the commercial
fungicide Thiram at the recommended dose (1
mL L-1). The concentrations used were
determined based on the in vitro test results.
To allow the emulsion between oil and water
we used Tween 80 (1 mL L-1) (Santos, 2018).
The seeds were disinfested in 2% sodium
hypochlorite solution for five minutes,
washed with sterile distilled water twice and
dried at room temperature. Afterwards they
were immersed for five minutes in the
different solutions (treatments). After drying
at room temperature, the artificial inoculation
was performed.
The inoculation was done depositing the seeds
on colonies of Fusarium verticillioides with 7
days of age. The seeds and the fungal colonies
stayed for 32 hours in a B.O.D (Biochemical
Oxygen Demand) type greenhouse at 27 ±
2°C, with a 12-hour photoperiod (Ramos et
al., 2014).
After the treatment and inoculation, the sanity
test of seeds was performed by the filter paper
method with freezing (Limonard, 1966). Six
hundred seeds of the hybrid (100 per
treatment) were used, distributed in Petri
dishes of 14 cm. Ten seeds were placed
equidistantly on each plate, on a triple layer of
filter paper previously moistened in sterile
distilled water, and incubated initially for 24
hours at 27 ± C with 12-hour photoperiod.
After this period, they were subjected to
freezing (-20° C) for 24 hours, and then
returned to the incubator for another five
days.
After incubation, the seeds were evaluated
individually, using a stereoscopic microscope
for the quantification of the seeds infected by
Fusarium verticilioides (Sacc.) Nirenberg.
The results were expressed as percentage of
infected seeds.
Statistics
To verify the effect of oil concentration on
fungal growth, we used quadratic plateau
regressions model for in vitro experimental
data and linear model for in vivo experiment
data. Regressions were performed using R
Core Team 3.5.1 software.
Due to the lack of variance in the results of
some treatments, the data were analyzed by
applying non-parametric tests. The difference
between treatments was verified by applying
the Mann-Whitney (Tukey non-parametric)
multiple comparisons. Differences with a
probability value below 5% were significant.
The analyses were performed using Past 3.12
(Hammer, Harper and Ryan, 2001).
Results and Discussion
Effect of palmarosa essential oil on
mycelial growth of Fusarium verticillioides
in vitro
All tested concentrations of palmarosa
essential oil inhibited mycelial growth of
Fusarium verticillioides. Inhibition
percentages increased significantly with the
concentrations tested until reaching the
maximum value (PGI = 100%) at the highest
concentration (0.2%) (Fig. 1A). The rate of
mycelial growth decreased with increasing
concentration of palmarosa oil. The minimum
value occurred (IMGS = 0 cm day-1) also in
the highest concentration (0.2%) (Fig. 1B).
According to the literature, the monoterpene
Geraniol is the major constituent of palmarosa
soil, in addition to other chemical components
such as geranial, linalool, thymol, limonene,
α-felandren, ocimene, germacrene-D and
isomentol that contribute to its potent
antifungal activity (Scherer et al., 2009,
Kalagatur et al., 2018). The mechanisms of
action of essential oil include lipid
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peroxidation, inhibition of ergosterol
biosynthesis and increase of reactive oxygen
species (ROS), which cause permeability of
cell membranes promoting loss of essential
molecules and affecting vital processes that
trigger the process of cell death by apoptosis
(Kalagatur et al., 2018).
Nascimento, Vieira, and Kronka (2016) found
similar inhibition results on Fusarium solani
f. sp. glycines at concentrations ranging from
1,000 to 8,000 μL L-1 (0.1 to 0.8%), under in
vitro conditions. The oil also inhibited
Sclerotium rolfsii, showing maximum
inhibition of mycelial growth in the
concentrations 500, 1000 and 1500 ppm
(0.05, 0.1 and 0.15%) (Guerra et al., 2015).
And, Khan and Ahmad (2012) testing the
control of Aspergillus fumigatus, obtained
maximum inhibition of 98.36% in the highest
concentration tested (0.32%). Although it is a
small variation, it suggests that palmarosa
essential oil may exert different antimicrobial
activity depending on the microorganism
studied, which justifies the investigation of its
minimum inhibitory concentration in other
phytopathogenic species.
Essential oil from other plant species also
significantly inhibited F. verticillioides. For
example, basil oil (Ocimum basilicum L.) lead
to total inhibition of fungal growth in the
concentration of 5 μL mL-1 (0.5%)
(Dambolena et al., 2010).
Using cinnamon oil (Cinnamomum spp.) at
concentration of 60 μL L-1 (0.06%), Xing et
al., (2014) found maximum mycelial growth,
while Bomfim et al., (2015) using rosemary
oil (Rosmarinus officinalis L.) obtained
maximum inhibition of 79.3% and 600 μg
mL-1 (0.06%).
To understand the potential of palmarosa
essential oil as a fungicide on F.
verticilioides, we compared its fungitoxic
effect with the effect of a commercial
synthetic fungicide. We found a stronger
inhibition effect of the essential oil
concerning the fungicide in the highest
concentration tested (Fig. 2), suggesting that
under in vitro conditions the commercial
synthetic fungicide can be replaced by the
essential oil.
The microbial control promoted by the
essential oils occurs through the synergism or
antagonism between several of its constituents
(Bagamboula; Uyttendaele, Debevere, 2004;
Russo et al., 2013) that act through different
mechanisms of action in several targets at the
same time (Abdel-Kader, El-Mougy and
Lashin, 2012; Hoyos et al., 2012). These
characteristics confer advantages over the
synthetic fungicide since they decrease the
phytopathogen resistance (Feng; Zheng,
2007).
Effect of palmarosa essential oil on
mycelial growth of Fusarium verticillioides
in maize seeds
Using maize seeds, the essential oil of
palmarosa exerted significant inhibitory effect
on F. verticillioides from the concentration of
3%. Levels higher than 3% reduced the seed
infection up to 21% at the highest
concentration (6%) (Fig. 3). The model
regression estimated a total reduction of
infected seeds at a concentration of 7.12%.
The present study results showed that
biologically active compounds present in
palmarosa oil promote a significant antifungal
effect on the mycelial growth of F.
verticillioides under in vitro conditions and in
the treatment of seeds. When using the oil at
the concentration of 0.2% we obtained total
inhibition of mycelial growth under in vitro
conditions. However, in seed treatment, a
higher concentration is required to obtain
significant inhibition of the fungus (<21% of
infected seeds).
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489
Fig. 1A Effect of different concentrations of palmarosa essential oil on the mycelial growth of
Fusarium verticillioides. 1B. Effect of different concentrations of palmarosa essential oil on the
mycelial growth speed of Fusarium verticillioides
**p<0.01; ***p<0.001; ns: not significant
Fig.2 Inhibition of mycelial growth of Fusarium verticillioides in the different concentrations of
palmarosa essential oil and the control treatments
Superscript concentrations with the same letter were not significantly different from each other by the Mann-
Whitney test (p>0.05)
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490
Fig.3 Effect of different concentrations of palmarosa essential oil on the incidence of
infected seeds by Fusarium verticillioides
***p<0.001
Fig.4 Percentage of infected seed by Fusarium verticillioides after the treatment with the
different concentrations of palmarosa essential oil and the control treatments
Superscript concentrations with the same letter were not significantly different from each other by the Mann-
Whitney test (p>0.05)
Fandohan et al. (2004) found significative
results of the effect of lemon grass essential
oil (Cymbopogon citratus) on the in vitro and
corn seeds control of F. verticillioides. Under
in vitro conditions, lemon grass oil totally
inhibited mycelial growth from the
concentration of 1.3 μL mL-1 (0.13%).
However, in the seed treatment it was also
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491
necessary to increase the concentration so that
there was a reduction in the percentage of
infected seeds.
The essential oil of palmarosa was more
efficient reducing seed infection than the
fungicide Thiram above 3% of concentration
(Figure 4). Despite needing a higher
concentration of oil to have a significant
reduction in infected seeds concerning the
Thiram, the use of a natural and healthy
product might be a better alternative to
control the fungus. However, minimum
recommended concentrations should be
considered to avoid toxicity in humans and
the environment.
According to Isman (2000), oils with the best
antifungal activities are toxic at high
concentrations. To test their constituents alone
is suggested, since the action of different
compounds present in the oil at high dosages
may be responsible for their toxicity.
Palmarosa oil, as well, is safe for human
health when used in low concentrations
(Sinha et al., 2014).
There is a growing interest in alternative
products to chemical pesticides because of
their high toxicity to humans and the
environment. Therefore, there is a demand
motivated by different priorities, such as
health benefits, food security and
environmental sustainability (Abdel-Kader,
El-Mougy and Lashin, 2012).
Our results can be used for the formulation of
natural defenses based on palmarosa essential
oil, to implementation in agroecological
crops, promoting the reduction of
environmental impacts caused by the
exclusive use of chemical pesticides.
However, it is important to establish safe
concentrations, respecting the oil toxicity
limit.
Palmarosa essential oil (Cymbopogon
martinii) totally inhibited the mycelial growth
of Fusarium verticillioides under in vitro
conditions from 0.2% concentration. In the
treatment of hybrid corn seeds AG 1051,
significantly reduced the percentage of
infected seeds from 3% of oil concentration.
The essential oil showed a higher effect than
the obtained by the commercial fungicide
Thiram.
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How to cite this article:
Kevison Romulo da Silva França, Alda Leaby dos Santos Xavier, Flavia Mota de Figueredo
Alves,Tiago Silva Lima, Ionaly Gomes de Araújo, Lídia Pinheiro da Nóbrega, Antônio Hugo
Costa Nascimento, Antônio Francisco de Mendonça Júnior, Ana Paula Medeiros dos Santos
Rodrigues, Antônio Fernandes de Almeida and Tiago Augusto Lima Cardoso. 2019. Control of
Fusarium verticillioides using Palmarosa essential oil (Cymbopogon martinii).
Int.J.Curr.Microbiol.App.Sci. 8(05): 484-494. doi: https://doi.org/10.20546/ijcmas.2019.805.057
... Study Design: The experiments comprised completely randomized designs: Seven treatments with five replicates on in vitro test; and four treatments with five replicates each, on in vivo test. Lima et al.; JEAI, 39(3): 1-11, 2019; Article no.JEAI.50072 2 Place and Duration of Study: The work was carried out at Methodology: In the in vitro test, the essential oil was incorporated into the PDA (Potato-Dextrose-Agar) culture medium. ...
... Using the essential oil of other plant species on control of F.verticillioides and Macrophomina spp., other authors obtained significant inhibition results. For example, the total inhibition of F. verticillioides was achieved by França et al. [39], Yamamoto-Ribeiro et al. [40] and Bonfin et al. [41] using the Palmarosa (Cymbopogon martinii), Ginger (Zingiber officinale) and Rosemary (Rosmarinus officinalis L.) essential oils at concentrations of 0.2%, 2.500 µg ml -1 (0.25%) and 150 µg ml -1 (0.015%), respectively. On control of M. phaseolina, Khaledi et al. [42] and Ugulino et al. [43] found maximum inhibition using Peppermint (Mentha piperita L.) and Alecrim-da-chapada (Lippia gracilis) at concentrations of 2.000 ppm (0.2%) and 0.4 to 1.0%, respectively. ...
... The present study showed that concentrations of essential oil used in seed treatment were not enough to significantly reduce the percentage of seeds infected by F. verticillioides. This result agreed with França et al. [39] who used the essential oil of Palmarosa (Cymbopogon martinii) on F. verticillioides in vitro and in the treatment of AG1051 maize seeds. Under in vitro conditions, the total inhibition was achieved from the 0.2% concentration, on the other hand, in the treatment of seeds there was significant reduction in the percentage of seeds infected by the fungus from the 3.0% concentration. ...
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Aims: This study evaluates the inhibitory potential of the clove essential oil (Syzygium aromaticum L.) on phytopathogenic fungi in vitro and on maize seeds. Study Design: The experiments comprised completely randomized designs: Seven treatments with five replicates on in vitro test; and four treatments with five replicates each, on in vivo test. Place and Duration of Study: The work was carried out at the Center for Agrifood Science and Technology of the Federal University of Campina Grande, Pombal, Brazil, from April to May 2018. Methodology: In the in vitro test, the essential oil was incorporated into the PDA (Potato-Dextrose-Agar) culture medium. The treatments comprised five concentrations of the oil (0.0125, 0.025, 0.05, 0.1, and 0.2%), a negative control (0.0%), and a positive control (Tiram). Plates were inoculated with the tested fungi, Fusarium verticillioides, Macrophomina phaseolina, and Macrophomina pseudophaseolina, then incubated for seven days at 27±2°C. The percentage of mycelial growth inhibition (PGI) and mycelial growth rate index (MGRI) were estimated. In the in vivo test, maize seeds (AG1051 hybrid) were treated with the essential oil on concentrations equal or superior to the minimum inhibitory concentration found in the in vitro test, besides the negative and positive controls. The artificial inoculation was carried out in fungi colonies for 32 hours and the seed sanity test was performed. The percentage of seeds infected by the fungus was evaluated after seven days. Results: In vitro conditions, clove oil totally inhibited the mycelial growth of F. verticillioides, M. phaseolina and M. pseudophaseolina at concentrations of 0.05, 0.1 and 0.1%, respectively. At 0.2% concentration significantly reduced the incidence of colonies of fungi M. phaseolina and M. pseudophaseolina in hybrid corn seeds AG 1051. Conclusion: The clove essential oil had a fungitoxic effect on the phytopathogens evaluated, under in vitro and in the treatment of maize seeds.
... The palmarosa EO has an antifungal activity well documented in the literature, and its biological activity has been studied in the last years. The main constituents of palmarosa EO are geraniol (82%), geranyl acetate (9%), linalool (2%), and trans-β-ocimene (1%), whereas geraniol is the main constituent associated with its antimicrobial activity [47]. The use of palmarosa EO presents promising results in the control of phytopathogens [23]. ...
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