H orizon Nexus Journal | Vol . 0 3 | Núm . 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 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/ )
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 91 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 92 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 94 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 95 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 96 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 97 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 98 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 99 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
Horizon Nexus Journal Horizon Nexus Journal | Vol.0 3 | Núm 0 2 | Abr Jun | 202 5 | www.horizonnexusjournal.editorialdoso.com 100 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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