In vitro evaluation with essential oils in watermelon (citrullus lanatus) and
its biological optimum, Guayas province.
Evaluación in vitro con aceites esenciales en sandia (citrullus lanatus) y su optimo biológico,
provincia del Guayas
Baque-Bustamante Wilmer
Master's Degree, Universidad Agraria del
Ecuador, Faculty of Agricultural
Sciences, Guayaquil, Ecuador
abarreto@uagraria.edu.ec
https://orcid.org/0000-0002-0089-1419
Barreto-Macias Arnaldo
Master's Degree, Universidad Agraria del
Ecuador, Faculty of Agricultural
Sciences, Guayaquil, Ecuador
abarreto@uagraria.edu.ec
https://orcid.org/0000-0002-0089-1419
Viñanzaca-Arellano Mariana
Master's Degree, Universidad Agraria del
Ecuador, Faculty of Agricultural
Sciences, Guayaquil, Ecuador
vinanzaca.arellano@uagraria.edu.ec,
https://orcid.org/0000-0002-7525-7113
Abstract
The purpose of this study was to evaluate in vitro the essential oils of Equisetum arvence and Urtica
dioica as fungicides at different concentrations on phytopathogenic fungi in watermelon. Samples were
collected in Empalme, Balzar and Pedro Carbo, in established crops that presented diseases. These
samples were taken to the phytopathology laboratory of the Agrarian University of Ecuador where the
samples were identified and isolated, duly disinfected in Petri boxes with PDA culture medium, and the
different concentrations were applied for each treatment. For this research work, two statistical designs
were used, under controlled conditions (laboratory), the complete randomized design with factorial
arrangement (A*B*C) 3 replicates, consisting of 3 pathogens, 3 doses, 2 treatments with 3 replicates,
and for the trial conducted with watermelon seedlings, the complete randomized design (CRD) was
used, which consisted of 3 phytopathogens (treatments) and 10 plants for each pathogen including the
control (replicates). According to the statistics, the treatment with the highest percentage of inhibition
was Urtica dioica essential oil with 36.19% and a concentration of 300 microliters with 34.38%. To see
the effect of the treatment on the watermelon seedlings, pathogenicity tests were carried out, where the
plants were inoculated with the pathogens found, then the best treatment was applied on the seedlings,
which resulted that the pathogen Curvularia sp and Colletotrichum sp had less severity with 2.7%.
abarreto@uagraria.edu.ec
http://centrosuragraria.com/index.php/revista, Published by: Edwards Deming Institute,
Quito - Ecuador, January - March vol. 1. Num. 12, 2022, This work is licensed under a
Creative Commons License, Attribution-NonCommercial-ShareAlike 4.0 International.
https://creativecommons.org/licenses/by-nc-sa/4.0/deed.es
Received Febrary 20, 2021
Approved: april, 12, 2021
74
Keywords: Equisetum arvence, oleomacerate, pathogens, watermelon, Urtica dioica
Resumen
El presente estudio tuvo como finalidad evaluar de forma in vitro los aceites esenciales de Equisetum
arvence y Urtica dioica como fungicida en distintas concentraciones sobre hongos fitopatógenos en
sandia. Las muestras fueron recolectadas En el Empalme, Balzar y Pedro Carbo, en cultivos
establecidos que presentaron enfermedades, dichas muestras se llevaron al laboratorio de fitopatología
de la Universidad Agraria del Ecuador donde se identificaron y se aislaron las muestras debidamente
desinfectadas en cajas Petri con medio de cultivo PDA, y se aplicó las diferentes concentraciones por
cada tratamiento. Para este trabajo de investigación se emplearon dos diseños estadísticos, bajo
condiciones controladas (laboratorio), se realizó el diseño completo al azar con arreglo factorial
(A*B*C) 3 repeticiones, que consta de 3 patógenos, 3 dosis, 2 tratamientos con 3 repeticiones, y para
el ensayo realizado con plántulas de sandía, se utilizó el diseño completo al azar (DCA), el cual constó
de 3 fitopatógenos (tratamientos) y 10 plantas por cada patógenos incluyendo las del testigo
(repeticiones). Según la estadística se reflejó que el tratamiento que tuvo mayor porcentaje de inhibición
fue el aceite esencial de Urtica dioica con el 36,19% y una concentración de 300 microlitros con
34,38%. Para ver el efecto del tratamiento en las plántulas de sandía se realizaron pruebas de
patogenicidad, donde se inocularon a las plantas con los patógenos encontrados, luego se aplicó el mejor
tratamiento sobre las plántulas, las cuales dieron como resultado que el patógeno de Curvularia sp y
Colletotrichum sp tuvieron menos afectación de severidad con un 2,7%.
Palabras clave: Equisetum arvence, oleomacerado, patógenos, sandía, Urtica dioica
Introduction
In Ecuador in 2010, 1908 ha of watermelon were planted with a production of 25818 tons. In addition,
about 378 ha were planted in association with other crops with a production of 383 t, with Guayas being
the province with the highest production 49 %, followed by Manabí 44 %, Los Ríos 3 % and Galápagos
1 % (Beltrán, 2015, p. 6).
Watermelon has been identified as a nutritious product for human consumption so it provides nutrients
such as lycopene, it also provides few calories, vitamins and minerals so it is good for health (Sylvain
and Enoch, 2019, p. 9).
Diseases in cucurbits are caused by microorganisms (fungi, bacteria, viruses, etc.) that are dispersed
mainly through water, air, insects and seeds. These microorganisms need certain environmental
conditions to develop and cause damage to plants. Among the main diseases affecting the Cucurbitaceae
family are leaf spot (Curvularia spp), Fusarium (Fusarium oxysporum), Alternaria (Alternaria
cucumerina) (González, et al., 2010).
One of the controls used to prevent fungal diseases is the use of natural compounds such as essential
oils, which are acquired from different plant organs such as flowers, roots, leaves, stems, fruits and
seeds, which have properties that are used to prevent or control the occurrence of diseases in plants.
These can be obtained by pressure, fermentation, or extraction, but the most common method for
commercial production is steam distillation (Mendoza, 2017).
Baque-Bustamante, Barreto-Macias, Viñanzaca-Arellano , 2022
January - March vol. 1. Num. 12 2022
One of the most important decisions in the phytosanitary management of the crop is the control of pests
and diseases to reduce the incidence and severity of pathogens, monitoring and analysis. On the other
hand, the analysis of statistical variables Analysis of Variance, Fisher's test and Tukey's comparative
test is important for the rational and economic use of essential oils has acquired special importance
within the technology for crop production. The present study will be carried out in (“SPSS Statistics -
Overview | IBM,” 2020), (“Herramientas estadísticas, de análisis de datos y de mejora de procesos |
Minitab,” 2020)
On the other hand, crop production is the result of a series of activities that transform inputs into
performance. Therefore, a production function represents the amount of product that could be obtained
using different doses or amounts of inputs, in this case oils for pathogen control. The most commonly
used response functions to explain plant response to growth inhibitors: quadratic, cubic, Cobb Douglas,
etc. Mention should also be made of the discontinuous rectilinear model approach, which also allows
economic interpretations. However, the most commonly used are the quadratic or second degree
functions, describing a curvilinear relationship between the expected production and the amount of
essential oil applied.
Materials and methods
For this type of study we will use linear regression, which is basically the simplest design and is used
when the experimental units are homogeneous or when the variation between them is very small; such
is the case of laboratory experiments, greenhouses, etc. where environmental conditions are controlled.
This is a test with only one classification criterion (Segura, 2000)
Experimental design is the arrangement of experimental units used to control experimental error by
adapting to the treatment. There are several arrangements in the literature aimed at controlling
experimental error, and there is a natural tendency to design experiments based on existing designs.
However, it is a more appropriate approach to develop an experimental design that meets the needs of
the ongoing experiments.(Kuehl, 2001)
Independent Variable
Evaluation of essential oils, with the concentrations of 100, 200, 300 μL in each experimental unit
(Pathogens).
Dependent Variable
Response of watermelon crop to the application of essential oils.
Variables To Be Evaluated
Mycelial growth of pathogens (%):
It was performed with the help of a millimeter ruler, the diameter of the colonies was measured at
intervals of 24 hours during 35 7 days. The formula for measuring mycelial inhibition growth according
to Sandoval (2016), is:
76
!"# $ %
&
#'()* +(,%-(.-/01 2 )'()* +(,%-'3-34/(5-1
6
)'()/4/(5-1%+(,%-(.-/01
%7%899
Pathogenicity Tests:
For these tests, 10 plants were inoculated for each pathogen (Fusarium sp. was inoculated by spraying
directly to the soil, while Curvularia sp. and Colletotrichum sp. were inoculated by foliar spraying).
These pathogenicity tests are effective and rapid since they facilitate the detection of the infectious
agent.
Symptom Assessment (%):
To determine the effect of diseases on plants, the percentage of severity was calculated using the
formula according to (Barea, 2006).
: $
;'(3%+(%-(</+1%=(0(-3,%3>()-3+1
-1-3,%+(%-(</+1.
%7%899
Table 1. Treatments
Viñanzaca, 2021
N. Treatments
Pathogens (B)
Dose( μL/ml) (C)
1
Fusarium sp.
100 μL
2
Fusarium sp.
200 μL
3
Fusarium sp.
300 μL
4
Fusarium sp.
100 μL
5
Fusarium sp.
200 μL
6
Fusarium sp.
300 μL
7
Colletotrichum sp.
100 μL
8
Colletotrichum sp.
200 μL
9
Colletotrichum sp.
300 μL
10
Colletotrichum sp.
100 μL
11
Colletotrichum sp.
200 μL
12
Colletotrichum sp.
300 μL
13
Curvularia sp
100 μL
14
Curvularia sp
200 μL
15
Curvularia sp
300 μL
16
Curvularia sp
100 μL
17
Curvularia sp
200 μL
18
P. cubensis
300 μL
19
No application
Baque-Bustamante, Barreto-Macias, Viñanzaca-Arellano , 2022
January - March vol. 1. Num. 12 2022
Table 2. ANDEVA analysis for laboratory
Viñanzaca, 2021
For the seedling trial, a complete randomized design (CRD) will be used, consisting of 3
phytopathogens (treatments) and 10 plants for each pathogen, including the control (replicates).
Table 3. ANDEVA analysis scheme for testing
Viñanzaca, 2021
Result
Isolation and identification of phytopathogenic agents associated with the watermelon crop Citrullus
lanatus L. Plant material (leaves, fruits, roots) was collected from established watermelon crops
showing symptoms of phytopathogenic agents by cluster or area sampling. Samples were collected in
the cantons of El Empalme, Balzar and Pedro Carbo. These samples were identified and isolated in the
Phytopathology laboratory of the Agrarian University of Ecuador. The phytopathogenic agents found
in the samples of vegetative material.
Model Summary
S R
2
R
2
(adjusted) R
2
(prediction)
0.348807 99.84% 99.77% 99.64%
Biological Optimum. To estimate it, it is necessary to calculate the dose of oils in microliters to
generate the maximum physical or biological production. In this case, we take the function obtained
and find the first order derivative which is also equivalent to the marginal product of the function
resulting in:
Source of variation
Formula
Development
g.l
A (Essential oils)
(A -1)
(2 -1)
1
B (Dosage)
(B - 1)
(3 - 1)
2
C (Pathogenic)
(C - 1)
(3 - 1)
2
AXB
(A - 1)(B -1)
(2 - 1) (3 - 1)
2
AXC
(A - 1)(C -1)
(2 - 1) (3 - 1)
2
BXC
(B - 1)(C -1)
(3 - 1) (3 - 1)
4
AXBXC
(A - 1)(B -1)(C - 1)
(2 - 1) (3 - 1)(3 - 1)
4
Experimental error
(T - 1) (R - 1)
(18 - 1) (3- 1)
34
Total
(N - 1)
(54 - 1)
53
Source of variation
Formula
Development
g.l
Treatments
(T - 1)
(4 - 1)
3
Experimental error
T (R - 1)
4 (10 - 1)
36
Total
(T * R) - 1
(4 * 10) - 1
39
78
%Inhibition = 30.6574 + 5.5352 Oils_T1 + 3.7204 Dose_300+ 1.7926 Pathogens_Curvularia sp. +
0.5870 Oils*Dose_T1 300 - 0.1630 Oils*Pathogens_T1 Curvularia sp.
+ 1.2630 Dose*Dose*Pathogens_300 Curvularia sp. + 2.2407 Oils*Dose*Dose*Pathogens_T1 300
Curvularia sp.
Evaluation of fungicide effectiveness on watermelon plants inoculated with pathogens.
To evaluate the effectiveness of the fungicide, watermelon plants of 7 weeks of age were used for the
test, inoculating the pathogen and showing the symptoms of each disease to later apply the dosage with
the best results in the laboratory, the formula was used to calculate the severity according to (Barea,
2006).
Severity of fungi (%) The data collected in the trial with watermelon seedlings of 7 weeks of age, the
oil of Urtica dioica was applied especially with the dose in 300 49 microliters where it caused the results
shown in table 10 which shows all averages, a coefficient of variation of 28.47% was obtained and
according to the variance if significance was found, between treatment the highest average of severity
category was obtained by the plant inoculated with Fusarium with 4,It should be noted that the
watermelon variety used has a property of tolerance to the pathogen, while Colletotrichum sp with 2.7%
equivalent on the severity scale has 25% of affectation, that is, it shows resistance to the pathogen since
the genetic material implemented also shows tolerance to this disease. It should be noted
It should be noted that the laboratory test indicates that Curvularia sp has a percentage of 32.42 in
PCIM, since the laboratory has controlled environmental conditions.
Table 4. Percentage of severity in seedlings.
Treatments
Stockings
n
E.E
T1 (Fusarium sp)
4.8
10
0.27
A
T2 (Colletotrichum sp)
2.7
10
0.27
B
T3 (Curvularia sp)
2.7
10
0.27
B
T4 (Witness)
1.9
10
0.27
B
Means with a common letter are not significantly different (p > 0.05).
The present research work evaluated the in vitro effect of essential oils of Equisetum arvense and Urtica
dioica for the management of phytopathogenic fungi in Citrullus lanatus L. Essential oils stand out for
their multiple possibilities of application in agriculture, thus providing an environmentally friendly
alternative, according to (Pino and Sanchez, 2011).
The study conducted by Mendoza (2017), showed that essential oils obtained from dehydrated leaves,
have better results since the plant component is in excellent condition at the time of extracting the oil,
it is a method currently used to control pests and diseases in order to minimize the incidence of diseases
that occur daily in crops, For this reason, the extraction of essential oils as a treatment limits the use of
agrochemicals, which bring with them the consequences of excessive use. For this reason, we agree
with Hernández (2013), who indicates in a research he carried out, applying essential oils to control
diseases and pests, since with this new alternative he seeks not to alter the environment.
Baque-Bustamante, Barreto-Macias, Viñanzaca-Arellano , 2022
January - March vol. 1. Num. 12 2022
After having carried out the analysis and interpretation of the mycelial inhibition growth data obtained
using the PCIM formula presented by Sandoval (2016), thus determining that the highest averages were
those of Treatment 1 (Urtica dioica) with 36.19%, since the nettle Urtica dioica contains properties
among them is silicon which is used as a fungicide since it possesses beneficial properties that can be
used in organic farming and to the defense of plants, so it agrees with Abad (2011), 51 in a research
where it indicates the properties that Nettle possesses as a product or fungicidal substance.
The percentage of severity was also evaluated in watermelon seedlings of 6 weeks of age, where it could
be deduced that essential oils do have fungicidal effect as indicated by Flores (2017), in a research
where he applied essential oils to control anthracnose in cucurbits. Inoculations of isolated diseases
were carried out on seedlings, and the essential oil U. dioica was applied, which resulted in a higher
inhibitory percentage, thus agreeing with the study conducted by Duarte (2013), where he mentions the
use of oils to combat Fusarium in inoculated seedlings. According to the research of the aforementioned
authors, the hypothesis that essential oils reduce the incidence of phytopathogenic fungi in watermelon
Citrillus lanatus is accepted.
Conclusions
Once the in vitro experimental research project was completed, where essential oils of Equisetum
arvence and Urtica dioica were evaluated in different concentrations, after the analysis study it was
concluded that when evaluating the inhibitory percentage on phytopathogenic fungi found in El
Empalme, Balzar and Pedro Carbo, the pathogens Colletotrichum sp, Fusarium sp, Curvularia sp.
It was determined that the essential oil of U. dioica obtained the highest percentage of inhibition of
mycelial growth with 36.19% with a dose of 300 microliters, these doses were evaluated on
phytopathogenic fungi (Fusarium sp, Colletotrichum sp, Curvularia sp), which resulted that Curvularia
sp has a percentage of 32.45%, pathogen that was more susceptible to the treatment and the lowest
average is Colletotrichum sp with 28.21%. It was also evidenced that when evaluating the severity,
pathogenicity tests were carried out on watermelon seedlings of 7 weeks of age, which were affected
with the pathogens found, statistically it was found that Curvularia sp together with Colletotrichum sp
are resistant to the pathogen, while Fusarium sp was considerably affected with 25% of affectation
according to the scale proposed, therefore it is concluded that the use of essential oils does reduce the
incidence of phytopathogenic fungi in the watermelon crop Citrullus lanatus.
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