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ISSN:
3073
-
1275
90
Evaluation of Azotobacter vinelandii as a biostimulant in the
development of cowpea (Vigna unguiculata)
Yessenia
Sarango
-
Ortega
1
,
*
y
Viviana
Sánchez
-
Vásquez
2
.
1
Universidad Estatal de Milagro, Milagro, Provincia del Guayas,
Ecuador,091050
;
https://orcid.org/0000
-
0001
-
7042
-
0623
2
Universidad Estatal de Milagro, Milagro, Provincia d
el Guayas,
Ecuador,091050
;
https://orcid.org/0009
-
0006
-
6911
-
3646
;
vsanchezv@unemi.edu.ec
*
Correspondenc
e
:
yesseniabia_93@hotmail.com
https://doi.org/10.70881/hnj/v3/n2/62
Abstract:
The study evaluated the effect of a biostimulant based on
Azotobacter vinelandii
on the germination, development and flowering of
cowpea (
Vigna unguiculata
).
A
.
vinelandii
is a nitrogen
-
fixing bacterium
capable of improving plant growth. An experiment was designed with seven
treatments and three repetitions, applying different doses of the biostimulant
(8 g, 12 g and 24 g) in combina
tion with drinking water (T1) and pineapple
peel water (T2). Variables such as germination rate, plant height, number of
leaves and flowers, length of the root system and stem thickness were
analyzed. The results showed that the biostimulant had a positive
impact on
germination and growth, with the 24 g dose standing out as the most effective,
especially in the development of the root system and leaf production.
However, no significant differences were observed in the number of pods
produced. It is conclude
d that
Azotobacter vinelandii
is a viable alternative to
improve cowpea yield, contributing to a more sustainable agriculture by
reducing the use of chemical fertilizers.
Keywords:
B
iostimulant;
Azotobacter vinelandii
; plant growth
Resumen
:
El estudio evaluó el efecto de un bioestimulante basado en
Azotobacter vi
nelandii sobre la germinación, el desarrollo y la floración del
caupí (Vigna unguiculata). A. vinelandii es una bacteria fijadora de nitrógeno
capaz de mejorar el crecimiento de las plantas. Se diseñó un experimento
con siete tratamientos y tres repeticion
es, aplicando diferentes dosis del
bioestimulante (8 g, 12 g y 24 g) en combinación con agua potable (T1) y
agua de cáscara de piña (T2). Se analizaron variables como la tasa de
germinación, la altura de la planta, el número de hojas y flores, la longitud
del
sistema radicular y el grosor del tallo. Los resultados mostraron que el
bioestimulante tuvo un impacto positivo en la germinación y el crecimiento,
destacando la dosis de 24 g como la más eficaz, especialmente en el
desarrollo del sistema radicular y
la producción de hojas. Sin embargo, no se
observaron diferencias significativas en el número de vainas producidas. Se
concluye que Azotobacter vinelandii es una alternativa viable para mejorar el
rendimiento del caupí, contribuyendo a una agricultura más
sostenible al
reducir el uso de fertilizantes químicos.
Palabras cla
ves
:
Bioestimulante; Azotobacter vinelandii; crecimiento
vegetal
Cit
e
:
Sarango
-
Ortega, Y., &
Sánchez
-
Vásquez, V. (2025).
Evaluation of Azotobacter
vinelandii as a biostimulant in
the development of cowpea
(Vigna unguiculata).
Horizon
Nexus Journal
,
3
(2), 90
-
102.
https://doi.org/10.70881/
hnj/v3/n2/62
R
eceived
:
10
/
02
/20
25
Revised
:
20
/
04
/20
25
Accepted
:
25
/
04
/20
25
Published
:
30
/
04
/20
25
Copyright:
© 202
5
by the
authors. This article is an open
access article distributed
under the terms and
conditions of the
Creative
Commons Attribution
-
NonCommercial 4.0
International License (CC
BY
-
NC).
(
https://creativecommons.org/
licenses/by
-
nc/4.0/
)
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1.
Introduction
The cowpea bean (
Vigna unguiculata
) is a legume of high nutritional value and great
importance in agriculture in tropical and subtropical regions due to its tolerance to low
fertility conditions (Morales, 2023). However, its production faces s
ignificant limitations,
such as the restricted availability of nitrogen in the soil, low germination rates, and
adverse environmental factors that compromise its development and flowering
(Daconceição et al., 2022). These constraints underscore the need fo
r sustainable
strategies that optimize crop yields without relying on the intensive use of synthetic
fertilizers (Higuera & Avellaneda, 2020).
Biostimulants based on beneficial microorganisms have emerged as an ecological
alternative to improve agricultura
l productivity.
Azotobacter vinelandii
, an atmospheric
nitrogen
-
fixing bacterium, also produces phytohormones such as auxins, cytokinins, and
gibberellins, promoting plant growth (Martin del Campo et al., 2022). Its application in
crops has been shown to i
mprove nutrient absorption and increase plant biomass (Peña
et al., 2015). In this context, assessing the impact of
A. vinelandii
on cowpeas could be
an effective strategy for sustainably increasing its productivity.
Despite its potential benefits, the use
of
A. vinelandii
in cowpeas still lacks sufficient
experimental evidence. Previous research suggests that plant response to biofertilizers
depends on the concentration applied and soil characteristics, which highlights the need
to establish optimal doses
to maximize their positive effects (W. Escobar et al., 2017). It
has also been documented that different levels of biostimulants can influence different
stages of plant development, which justifies specific studies for each crop and
environment (Quintero e
t al., 2018).
The present study aims to determine the optimal concentration and type of suspension
of a biostimulant based on
A. vinelandii
to improve the germination, growth, and flowering
of cowpea beans (Lara et al., 2010). It is
hypothesized that the application of this
biostimulant in adequate doses will significantly increase morphological parameters such
as plant height, number of leaves, stem thickness, and flower and pod production
compared to a control group. To evaluate thi
s hypothesis, an experiment was designed
using three concentrations of the biostimulant (8 g, 12 g, and 24 g), dissolved in drinking
water (T1) and pineapple peel water (T2) (Mero, 2021), The latter as a source of
enzymes, organic compounds and bioactive m
etabolites that could favor the activity of
A. vinelandii
.
The main challenge of this research lies in the development of efficient methods to
optimize cowpea bean production without resorting to the excessive use of synthetic
fertilizers, whose negative e
nvironmental impact and high cost have been widely
documented (Daconceição et al., 2022) The application of nitrogen
-
fixing
microorganisms such as
A. vinelandii
represents a promising alternative to improve soil
fertility and reduce dependence on chemica
l inputs in agriculture (Huaman, 2021).
This analysis aims to produce evidence about the effectiveness of
A. vinelandii
as a
biostimulant in cowpeas, determining the ideal dose to enhance its influence on crop
yield. Promoting the use of natural biofertili
zers not only increases productivity in
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agriculture but also promotes the reduction of agrochemical use and the implementation
of sustainable agriculture models (Albarracin, 2024). The results achieved could be used
in the creation of more effective agrono
mic management strategies, favoring both small
-
scale producers and the preservation of the natural environment.
2. Material
and
Methods
The purpose of this study was to assess the impact of a biostimulant based on
A
.
vinelandii
on germination,
development, and flowering of cowpea (
Vigna unguiculata
).
To achieve this, an experiment was conducted under field conditions with different doses
of the biostimulant, evaluating its influence on the morphological and physiological
variables of the crop.
S
tudy Design
The present research is framed within an experimental study, with a quantitative and
explanatory approach, whose objective is to establish the causal relationship between
the application of the biostimulant and the growth of cowpea beans. For
this purpose, a
completely randomized experimental design was adopted, in which seven different
treatments were implemented, each replicated three times. The variables evaluated in
the study included germination speed, plant height, stem thickness, number
of leaves
and flowers, root system length, as well as the number of pods produced.
The test was carried out in Milagro, Ecuador, in soil with a sandy loam texture and a
climate marked by an average temperature of 25°C and a relative humidity of 80%.
Black
-
eyed pea seeds were used. The biostimulant used was
A
.
vinelandii
at doses of
8g, 12g, and 24g, purchased from the Asociación de Producción Industrial Licán
(ASOPROIL) mixed with drinking water (T1) and pineapple peel water (T2).
Inclusion and exclusion
criteria
To ensure the uniformity of the research, specific criteria were established for the choice
of biological material:
• Seeds of homogeneous size and without perceptible imperfections were chosen. The
plants analyzed had to have completed their ger
mination stage under normal conditions.
• Seeds showing signs of physical damage or disease before planting were removed. In
addition, seeds that did not germinate on schedule were discarded.
• Throughout the experiment, plants with irregularities in their
development or that were
impacted by pests and diseases not related to the research were discarded.
Procedure
The experimental procedure involved several fundamental phases. First, the seeds were
subjected to pretreatment by submerging them in solutions w
ith different doses of
biostimulant for 12 hours, and then manually sowing in plots arranged in three rows with
five holes each, with a depth of 3 cm, placing two seeds in each hole. Then, solutions of
the biostimulant, prepared in 8L of drinking water (T1
) and pineapple peel water (T2),
were used utilizing irrigation oriented to the base of the stem. Weekly applications were
carried out for six weeks to ensure uniform distribution. Additionally, to verify the quality
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93
and viability of the biostimulant (
A
.
v
inelandii
), a cell count was performed using the
Neubauer chamber method and microscopic observation at 400x.
Irrigation was drip irrigated for the first 15 days and then every other day, while weed
management was carried out by manual removal every 15 da
ys and the use of garlic
extract as a natural pest repellent. Finally, weekly evaluations of factors such as
germination rate, plant height, leaf width, stem thickness, number of leaves and flowers,
number of pods, and length of the root system were carrie
d out.
Data analysis
The data collected were grouped in matrices and examined by ANOVA to detect relevant
differences between treatments. To contrast the averages and establish which
biostimulant dose showed the best performance in each variable, Tukey's
multiple
comparisons test was used with a significance level of 0.05. Statistical analyses were
carried out using the MINITAB program.
3. Results
The evaluation of the findings made it possible to assess the impact of the biostimulant
based on
A
.
vinelandii
on the germination, development, and flowering of cowpea (
Vigna
unguiculata
), exposing the most significant findings concerning the variables analyzed.
Seed germination
The germination rate was generally high in most treatments, with small varia
tions. It was
observed that seeds treated with the biostimulant presented a higher germination
percentage compared to the control group. In particular, the 8g (D1) and 12g (D2) doses
in the T1 treatment achieved the highest number of germinated seeds (15 i
n total). On
the other hand, the 12g (D2) dose in treatment T2 showed the lowest germination rate
(11 germinated and 4 non
-
germinated seeds) (Figure 1).
Figure 1
Seed germination
14
15
15
14
14
11
14
0
2
4
6
8
10
12
14
16
T0D0
T1D1
T1D2
T1D3
T2D1
T2D2
T2D3
Seed Germination
Treatments
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Note.
Number of germinated seeds for each treatment
T0D0 (Control), T1 (Treatment 1
–
A.
vinelandii
+ Drinking water), T2 (Treatment 2
–
A. vinelandii
+ Pineapple peel water), D1 (Dose
1
-
8g), D2 (Dose 2
-
12g), D3 (Dose 3
-
24g).
Source:
Authors
Plant Height
Height measurements showed that the
biostimulant treatments promoted the
development of cowpeas, with notable differences between the doses used. The results
indicate that the greatest height was obtained in the 24 g (D3) dose of treatment 2 (29.78
cm), followed by the same dose in treatment
1 (26.86 cm). In contrast, the control
treatment and the 12g (D2) dose in treatment 2 recorded the smallest values (Figure 2).
The analysis of variance showed significant differences between treatments (p = 0.059),
with 6.46% of the variability attributab
le to the treatment and 41.90% to experimental
error. This suggests that the biostimulant has a positive effect on plant height, although
other factors also influence growth.
Figure 2
Effect of treatments on plant height.
Note.
Average plant height (cm)
under different treatments. T0D0 (Control), T1 (Treatment 1
–
A. vinelandii
+ Drinking water), T2 (Treatment 2
–
A. vinelandii
+ Pineapple peel water), D1
(Dose 1
-
8g), D2 (Dose 2
-
12g), D3 (Dose 3
-
24g). The highest height in T2D3 (29.78 cm) and
the low
est in T2D2 (16.89 cm).
Source:
Authors
Leaves width
Leaves width also showed considerable variations among the different treatments. The
plants that were treated with
A. vinelandii
presented wider leaves compared to the control
group, highlighting the do
se of 24g (D3) at T1 with an average of 4.49 cm (Figure 3). The
analysis of variance revealed a marginally significant difference between treatments (p
= 0.055), with 16.31% of the variability explained by the treatments. These findings
indicate that the b
iostimulant could improve leaf structure, favoring light uptake and thus
photosynthetic efficiency.
20,06
20,58
21,97
26,86
21,96
16,89
29,78
0
5
10
15
20
25
30
35
T0D0
T1D1
T1D2
T1D3
T2D1
T2D2
T2D3
Plant height (cm)
Treatments
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Figure 3
Variation in leaf width (cm) under different treatments
Note.
Average leaf width (cm) as a function of different treatments. T0D0
(Control), T1 (Treatment
1
–
A. vinelandii
+ Drinking water), T2 (Treatment 2
–
A. vinelandii
+ Pineapple peel water), D1
(Dose 1
-
8g), D2 (Dose 2
-
12g), D3 (Dose 3
-
24g). Source: Authors
Stem size
Regarding stem size, the T2 treatment recorded the high
est yield with the 24g dose (1.62
cm), followed by the T1 treatment with the 12g dose (1.47 cm), while the control group
showed the lowest results (Figure 4). The analysis of variance showed a significant
difference between treatments (p = 0.055), with 13.
36% of the variability presented by
the biostimulant. This suggests that
A. vinelandii
may contribute to stem thickening,
providing greater structural support to the plant.
Figure 4
Effect of the treatments on stem thickness
3,12
3,98
3,73
4,49
3,74
3,12
4,07
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
T0D0
T1D1
T1D2
T1D3
T2D1
T2D2
T2D3
Leaf width (cm)
Treatments
1,12
1,43
1,47
1,31
1,31
1,21
1,62
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
T0D0
T1D1
T1D2
T1D3
T2D1
T2D2
T2D3
Stem Thickness (cm)
Treatments
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Nota
.
Average stem thickne
ss (cm) in response to different treatments. T0D0 (Control), T1
(Treatment 1
–
A. vinelandii
+ Drinking water), T2 (Treatment 2
–
A. vinelandii
+ Pineapple peel
water), D1 (Dose 1
-
8g), D2 (Dose 2
-
12g), D3 (Dose 3
-
24g).
Source:
Authors
Leaf
development and flowering
The number of leaves showed a positive response to the biostimulant, treatment T1
with dose D3 presented the highest number of leaves, with an average of 13.57 leaves
per plant (Figure 5). The analysis of variance showed a signif
icant difference between
treatments (p = 0.067), with 2.56% of the variability explained by the treatments.
Figure 5
Variation in the average number of leaves under different treatments
Note.
The average number of leaves in plants subjected to
different treatments. T0D0 (Control),
T1 (Treatment 1
–
A. vinelandii
+ Drinking water), T2 (Treatment 2
–
A. vinelandii
+ Pineapple
peel water), D1 (Dose 1
-
8g), D2 (Dose 2
-
12g), D3 (Dose 3
-
24g).
Source:
Authors
Concerning flowering, significant diff
erences were observed among treatments,
highlighting the 24g dose at T2, with an average of 1.83 flowers per plant, indicating that
A. vinelandii
can promote higher floral production in cowpea (Figure 6). Analysis of
variance showed a marginally significan
t difference (p = 0.065), suggesting that the
biostimulant could stimulate floral production by enhancing nutrient uptake and
availability of bioactive compounds.
8,67
10,4
11,62
13,57
10,57
11,15
12,87
0
2
4
6
8
10
12
14
16
T0D0
T1D1
T1D2
T1D3
T2D1
T2D2
T2D3
Number of leaves (#)
Treatments
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Figure 6
Number of flowers in response to different treatments
Nota.
The
average number of flowers in plants subjected to different treatments. T0D0 (Control),
T1 (Treatment 1
–
A. vinelandii
+ Drinking water), T2 (Treatment 2
–
A. vinelandii
+ Pineapple
peel water), D1 (Dose 1
-
8g), D2 (Dose 2
-
12g), D3 (Dose 3
-
24g).
Source
:
Authors
Pod production and root development
In the analysis of the number of pods per plant, no significant differences were found
among treatments, since all presented similar values, with a maximum average of 0.13
pods per plant
in T1 and 2 in dose 3 (Figure 7).
Figure 7
Number of pods in response to different treatments
0,1
0,08
0,1
0,13
0,12
0,11
0,13
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
T0D0
T1D1
T1D2
T1D3
T2D1
T2D2
T2D3
Number of pods (#)
Treatments
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Note.
The average number of pods in plants subjected to different treatments. T0D0 (Control),
T1 (Treatment 1
–
A. vinelandii
+ Drinking water), T2 (
Treatment 2
–
A. vinelandii
+ Pineapple
peel water), D1 (Dose 1
-
8g), D2 (Dose 2
-
12g), D3 (Dose 3
-
24g).
Source:
Authors
The root system showed a favorable response to the biostimulant, with significant
differences among treatments. The greatest root l
ength was recorded in treatment T2
with dose D3, reaching an average of 10.44 cm, followed by T1 with dose 2 (9.71 cm),
while the control group presented the shortest roots (4.33 cm) (Figure 8). The analysis
of variance reflected significant differences be
tween treatments (p = 0.043), with 19.68%
of the variability, these results reinforce the hypothesis that
A. vinelandii
could improve
water and nutrient absorption capacity by stimulating root development.
Figure 8
Effect of treatments on the root system.
Note.
Average root system length (cm) in response to different treatments. T0D0 (Control), T1
(Treatment 1
–
A. vinelandii
+ Drinking water), T2 (Treatment 2
–
A. vinelandii
+ Pineapple peel
water), D1 (Dose 1
-
8g), D2 (Dose
2
-
12g), D3 (Dose 3
-
24g).
Source:
Authors
4. Discussion
The results obtained support the hypothesis that this microorganism promotes plant
growth by enhancing nutrient uptake and stimulating key physiological processes.
An increase in the
germination of seeds treated with the biostimulant was observed in
comparison with the control group, with greater effectiveness at the doses of 12g (D1)
and 18g (D2) in T1. This coincides with previous studies showing that
A. vinelandii
promotes germinati
on by producing phytohormones such as indoleacetic acid (IAA) and
gibberellins, which stimulate cell elongation and early root emergence (Escobar et al.,
2011). In addition, its ability to fix atmospheric nitrogen improves the availability of
essential nut
rients for initial plant development (Pérez et al., 2014).
4,33
8,68
9,71
9,56
9,36
9,61
10,44
0
2
4
6
8
10
12
T0D0
T1D1
T1D2
T1D3
T2D1
T2D2
T2D3
Root length (cm)
Treatments
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However, the lower germination observed at the 12 g dose in T2 could be related to
environmental factors or to the presence of metabolites in the pineapple water used in
this treatment, which could
affect seed viability.
In plant height, the biostimulant significantly favored the development of cowpea beans,
with a greater height at the 24 g dose at T2 (29.78 cm). The literature indicates that A.
vinelandii improves plant growth by producing siderop
hores, which facilitate iron uptake,
and volatile compounds that modulate the expression of genes related to plant
development (M. D. Sánchez, 2017).
Likewise, a positive effect on leaf width and stem thickness was also observed,
suggesting an improvement
in photosynthetic capacity and structural resistance of the
plant. These results are consistent with research indicating that
Azotobacter spp.
can
improve leaf morphology and water use efficiency by inducing the accumulation of
osmoprotectants such as prol
ine and trehalose (Pedraza et al., 2018). Although the
differences are significant, the variability in some treatments suggests that other factors
such as soil nutrient availability or interaction with native microorganisms may influence
growth.
The applic
ation of the biostimulant had an impact on flowering, with a higher number of
flowers at the 24 g dose at T2 (1.83 flowers/plant). This suggests that
A. vinelandii
stimulates the synthesis of phytohormones involved in flowering, such as cytokinins and
ethy
lene at low concentrations (Velasco et al., 2020). However, no significant differences
were found in pod production between treatments, indicating that the conversion of
flowers to fruit may depend on other factors such as pollination and climatic conditio
ns
(Quintero et al., 2018).
Previous studies report that legume yield response to biostimulants is highly variable and
depends on the interaction with soil microbiota and macronutrient availability (Escobar
et al., 2011) Therefore, it is possible that the
positive effect of the biostimulant on
flowering may not translate into a higher number of pods due to competition for resources
at later stages of development.
The most pronounced effect of the biostimulant is observed in the root system, with a
greater r
oot length at the 24 g dose at T2 (10.44 cm). This reinforces the hypothesis that
A. vinelandii
enhances nutrient and water uptake by stimulating root growth through the
production of growth regulators and phosphate solubilization (Ariza et al., 2020).
The
se findings coincide with research on other legumes, where it has been shown that
plant growth
-
promoting bacteria can increase root biomass by up to 30%, improving the
efficiency of water and nutrient uptake, which is crucial in soils with low fertility (S
antoyo
et al., 2023).
This analysis provides evidence of the benefit of
A. vinelandii
on cowpea development,
highlighting its capacity as a sustainable biofertilizer in the agricultural sector. However,
further studies are needed to enhance its effectivene
ss. Specifically, it is crucial to
analyze its interaction with the soil microbiota, since its performance is based on
coexistence and rivalry with indigenous microorganisms (J. M. Sánchez et al., 2022). It
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is also advisable to examine other agronomic elem
ents, such as fertilization and
pollination, since these can affect the final performance of the crop (Pérez
-
Pazos &
Sanchez
-
Lopez, 2017). Although an increase in flowering is noted, pod production does
not show notable variations, indicating the importanc
e of investigating the underlying
processes that influence the transformation of flowers into viable reproductive structures.
5. Conclusions
This research demonstrates the capacity of
A. vinelandii
as a biostimulant in cowpea
production, prevailing its ro
le in increasing plant growth through sustainable biological
processes. Its ability to capture atmospheric nitrogen and generate phytohormones
makes it a feasible option compared to artificial fertilizers, with direct advantages in
agricultural productivit
y and the reduction of the environmental effect linked to the use of
agrochemicals.
One of the most relevant contributions of this research is the identification of a positive
effect of the biostimulant on the growth and development parameters of cowpeas,
which
supports the importance of exploring beneficial microorganisms as allies in sustainable
agriculture. In addition, the findings suggest that the formulation and the medium in which
A. vinelandii
is applied may influence its effectiveness, opening the
door to future
research on the optimization of its use in different crops and soil and climatic conditions.
The promotion of root growth observed in this research is an indication that
A. vinelandii
could improve the efficiency of nutrient and water uptake
, which would be key in the
adaptation of crops to environmental stress conditions. From an applied perspective, the
results reinforce the need to promote agricultural practices that integrate beneficial
microorganisms as biotechnological tools accessible
to small and medium
-
sized
producers.
Therefore, this study establishes a basis for future research focused on the interaction
of
A. vinelandii
with the soil microbiota, its persistence in diverse agricultural systems,
and its influence on final crop produc
tion. The importance of understanding more deeply
the variables that control the transformation of flowers into viable reproductive structures,
together with the impact of the biostimulant on final yield, generates new possibilities for
refining its use in
modern agriculture.
Contributions
a
uthor:
conceptualization, YB
-
SO. and VLSV.; methodology, YB
-
SO.;
software, YB
-
SO.; validation, YB
-
SO.; formal analysis, YB
-
SO.; research, YB
-
SO. and
VLSV.; resources, YB
-
SO. and VLSV.; writing the original draft, YB
-
SO.;
drafting,
revising and editing, YB
-
SO.; visualization, YB
-
SO.; supervision, YB
-
SO. All authors
have read and accepted the published version of the manuscript
.
Funding:
This research has not received external funding.
Acknowledgments:
We thank all
those who collaborated in the conduct of this study,
providing technical and academic support. Their contribution was essential for the
development of this research.
Data availability statement:
Data are available upon request to the authors of
corresponde
nce: yesseniabia_93@hotmail.com
Conflict of interest:
The authors declare that they have no conflict of interest.
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