Pasture Science and other Crops

Effect of inoculation with beneficial microorganisms on agroproductive variables of Morus alba

0000-0002-5738-7838C. J. Bécquer^{1  *}  , Adelaida B. Puentes²  , Arachely Cabrera²  , María Hernández¹  , Ana Sánchez¹ 

¹Instituto de Investigaciones de Pastos y Forrajes, Estación Experimental de Sancti Spíritus, Cuba (MINAG-GEGAN)

²Universidad José Martí Pérez, Sancti Spíritus, Cuba

Abstract

A field experiment was carried out to evaluate the effect of the simple and combined inoculation of Azospirillum brasilense and Glomus cubense on agroproductive variables of Morus alba. An experimental design of control plots was used, with six treatments and ten replications. Conventional inoculation techniques were applied. The treatments consisted of EcoMic^®, INICA-8, INICA-8+ EcoMic^®, absolute control, control fertilized with organic matter and control chemically fertilized. The main stem length (cm), main stem diameter (cm), number of branches, number of leaves, dry weight of leaves (g/plant) were evaluated and the inoculation efficiency index was calculated on the basis of the dry weight of leaves (%).Simple ANOVA analysis and comparison of means by Fisher's LSD were performed. The digit count data were transformed by √x. In the main stem length, the chemically fertilized control highlighted due to its higher values (P <0.001) with respect to the rest of treatments, although it shared common letters with the control fertilized with organic matter at 45 (90.15 cm) and 75 days after sowing (119.50 cm), as well as with EcoMic^® + INICA-8, at 135 days after sowing (190.40 cm). Regarding the dry weight of leaves, the treatment inoculated with the combination of EcoMic^® + INICA-8 (78.51 g/plant) and the control fertilized with organic matter (76.79 g/plant) were higher to the rest (P <0.001) , and statistically similar to each other. It is concluded that the combined inoculation of EcoMic^® + INICA-8 exerted a higher effect on the studied variables. The variables that were most favored with the inoculation were the stem length and the number of branches. The simple inoculation of INICA-8 was inefficient in all variables.

Key words: Azospirillum brasilense; Glomus cubense; nutrition; mulberry

The cultivation of mulberry (Morus alba L.) is an option to guarantee a more balanced animal nutrition. It is a highly productive forage shrub that, although is native to Asia, has adapted excellently to the tropics (^{Benavides 1994}). However, its intensive exploitation requires high amounts of nutrients in the soil, especially nitrogen and potassium, of which it requires a continuous supply (^{Pentón et al. 2014}).

To avoid the continuous application of chemical fertilizers, the use of microbial inoculants constitutes an alternative in the search for resources that are friendlier to the environment. The action of soil microorganisms contributes to the sustainability of all ecosystems, when regulating the organic matter dynamics, the carbon sequestration from soil and the emission of greenhouse gases, modifying the physical structure of the soil and the water regime, thus improving the efficiency of nutrient acquisition (^{Singh et al. 2011}).

^{Plana et al. (2016)} point out that the use of arbuscular mycorrhizal fungi (AMF) leads to a symbiosis of the microorganism with the plant, which allows transporting the necessary nutrients for its metabolism and, at the same time, improves the chemical, physical and biological properties of the soil. It is known that the AMF not only make the extraction of nutrients from the soil more efficient, but also reduce their loss due to washing (^{Bender et al. 2015}).

Azospirillum brasilense is another of the microorganisms that are valued for their contribution to the promotion of plant growth. ^{Wing Ching-Jones et al. (2016)} showed that the application of different strains of Azospirillum in Cynodon nlemfuensis has a similar effect to the application of nitrogen fertilizer, in terms of the yield of aerial biomass.

When referring to the agronomic performance of mulberry in Cuba, where there is knowledge among farmers about the use of organic fertilizers and biofertilizers, ^{Martín (2010)} points out that it is possible to use other sources of fertilization in the country. Therefore, it would be necessary to study the performance of these sources and their combination with chemical fertilizers.

Due to the previous mentioned, the objective of this study was to evaluate the effect of simple and combined inoculation of Azospirillum brasilense and Glomus cubense in agroproductive variables of mulberry.

Materials and Methods

Location of the experiment. The experiment began on September 19, 2019 in an experimental plot belonging to the Estación Experimental de Pastos y Forrajes Sancti Spíritus, Cuba (21^o 53´ 00´´ north latitude and 79^o 21´ 25´´ west longitude, altitude 40 m o.s.l).

Plant material. Morus alba Linn (Moraceae), Yu-62 variety, from the germplasm bank of the Sancti Spíritus Experimental Station was evaluated.

Bacterial strain. The INICA-8 strain, from Azospirillum brasilense, provided by the Instituto de Investigaciones de Pastos y Forrajes (IIPF) was applied. According to the manufacturer's recommendations, the preparation was diluted in common water, in a ratio of 1:10. The inoculation was performed at the time of transplantation of mulberry seedlings, with an inoculum of 10⁹-10¹⁰ CFU /mL. A 12 L sprayer backpack was used, whose content was dropped on the soil around the seedling. When regulating the spout, each plant approximately received 125 mL of liquid inoculum (40 L/ha of pure inoculum).

Strain of arbuscular mycorrhizal fungi (AMF). The inoculation was performed using the EcoMic^® certified mycorrhizal inoculant, which contained 30 spores/g of substrate produced in the Departamento de Biofertilizantes y Nutrición from INCA. It was applied solid in the soil around the plant (15-20 g/plant), immediately after transplanting the seedling, with a dose equivalent to 50 kg/ha.

Fertilization with organic matter. A mixture of soil and organic matter (3: 1 ratio) was applied, in a dose equivalent to 30 t/ha, whose composition is shown in table 1.

Table 1 Chemical characteristics of soil and organic matter mixture 

K2O, (mg/100g)	P2O5, (mg/100g)	O.M, (%)	pH, H2O
17.10	10.90	5.29	6.90

Chemical fertilization. Phosphoric carrier (P₂O₅) was applied in doses of 30 g/plant, equivalent to 100 kg/ha, amount recommended for mulberry (^{Cifuentes and Sohn 1998}).

Agrotechnics of the experiment. It was performed from September 19, 2019 to April 15, 2020, in an area of 40 x 40 m, destined to mulberry sowing in seedlings to obtain green forage. A conventional soil preparation was carried out: plowing, medium harrow, light harrow, crossing, recrossing and furrowing. A 1 x 0.5 m frame was used, with a sowing density of 20,000 pl/ha, for a total of 3,200 plants in the experimental area. Root ball seedlings were used, which were sown at a depth of 10 cm. Irrigation (250 m³/ha) was applied three times in September (once during the transplanting of seedlings, and two immediately after inoculation). It was not irrigated in October because this month there was rainfalls that guaranteed sufficient soil moisture. However, it was irrigated three times in November, and twice in December, January, February, March, and April, respectively.

Evaluation of the climatic variables in the area and experimental period. The variables rainfalls (mm), temperature (^oC) and relative humidity (%) were evaluated. The data were provided by the Delegación Provincial del Instituto de Meteorología (INSMET) in Sancti Spíritus.

Basic agrochemical composition of the experimental soil. The soil in the experimental area (table 2) corresponded to the loose brown carbonate type (^{Hernández et al. 2015}), made of brown or slightly dark brown clay, with a slight reaction to HCL. It has some gravels on the A1 horizon, good superficial and internal drainage, moderately erodible.

Table 2 Basic characteristics of the experimental area soil 

Type of soil	P2O5, mg/100g	K2O mg/100g	OM,%	pH (KCl)
Loose brown carbonate	10.52	15.00	2.90	5.9

Experimental design and statistical analysis. An experimental design of control plots was used (^{Lerch 1977}), with six treatments and ten replications (table 3). The differences between means were determined by Fisher's LSD (^{Fernández et al. 2010}). The digit count data were transformed by√x. The statistical program StatGraphics Centurion XV (^{Anon, 2007}) was used.

Table 3 Treatments used in the experiment 

No.	Treatments
1	EcoMic^®
2	INICA-8
3	INICA-8+ EcoMic^®
4	Absolute control (AC)
5	Fertilized control with organic matter (FCOM)
6	Chemically fertized control (P2O5FC)

Evaluated variables. The variables length and the main stem diameter, number of branches and leaves and dry weight of leaves were measured.

To determine the main stem length (MST, cm), it was measured from the stem base to the apex, using a tape measure. Six evaluation moments were taken into account, days after sowing (DAS) (45, 75, 105, 135 and 165 DAS).

The main stem diameter (MSD, cm) was measured at the cutting time, 20 cm from the stem base, with a vernier.

To determine the dry weight of leaves (DWL, g), they were placed in a MEMMERT oven, at a temperature of 70 ^oC, for seven days. Subsequently, the weighing was performed on an OHAUS technical digital scale, with 1 kg capacity.

The inoculation efficiency index (IEI, %) was determined, according to the ^{Santillana et al. (2012)} formula:

[TeX:] IEI: [(Inoculated treatment- Absolute control)/Absolute control] x 100

Results and Discussion

Figure 1 shows the rainfalls during the period September/2019-April/2020. Abundant rainfalls characterized September and October 2019. However, in the period from December 2019 to April 2020 they were very low in December, January, February, March and April. In this interval, important phenological stages of the crop occurred, which coincided with a very low level of rainfalls.

Figure 1 Rainfalls from September 2019 to April 2020 

As shown in figure 2, the highest average temperatures in the experimental period occurred in September 2019 and April 2020, months in which the sown and cut of the experiment was made. The maximum temperatures reached their highest values also in these intervals, while the minimum temperatures were recorded in December 2019 and January 2020.

Figure 2 Average temperature from September 2019 to April 2020 

Figure 3 shows that the highest value of relative humidity was registered in October 2019, while the lowest was in April 2020. In the latter, the experiment cut was made.

Figure 3 Relative humidity from September 2019 to April 2020 

At 15 DAS, the INICA-8 treatment (51.90 cm) showed higher values (P <0.001) than the AC (40.30 cm) and EcoMic^® (44.40 cm), although it was equal to the FCOM (52.63 cm) and to EcoMic^® + INICA -8 (47.80 cm). However, it was lower than P₂O₅FC (62.30 cm) (figure 4).

At 45 DAS, P₂O₅FC (86.12 cm), as well as FCOM (90.15 cm) were higher (P <0.001) to the absolute control (67.06 cm), while they showed letters equal to the three inoculated treatments. The latter showed statistically equal values among themselves and with respect to the absolute control (figure 4).

At 75 DAS, the P₂O₅FC (141.40 cm) reached higher values (P <0.001) than the rest of the treatments, with the exception of FCOM (119.50 cm). EconoMic^® (118.40 cm), INICIA-8 (111.05 cm) and EconoMic^® + INICA-8 (97.79 cm) were statistically equal, but only EcoMic^® and INICA-8 were higher to AC (figure 4).

At 105 DAS, P₂O₅FC (193.90 cm) showed higher values (P <0.001) than the rest of the treatments, while EcoMic^® + INICA-8 (167.10 cm) and FCOM (158.50 cm), although they shared common letters with INICA-8 (154.60 cm), were higher to EcoMic^® (140.30 cm) and AC (130.60 cm) (figure 4).

At 135 DAS, P₂O₅FC (206.20 cm) showed higher values (P <0.001) than the rest of the treatments, except for EcoMic^® + INICA-8 (190.40 cm), with it was statistically similar. The latter, in turn, shared common letters with EcoMic^® (182.33 cm), and was higher to INICA-8 (167.00 cm), FCOM (164.70 cm) and AC (156.33 cm) (figure 4).

At the time of cutting (165 DAS), EcoMic^® + INICA-8 (213.33 cm) showed superiority (P <0.001) in relation to INICA-8 (186.90 cm) and AC (174.90 cm), although it shared common letters with EcoMic^® (196.00 cm) and with FCOM (195.90 cm), being only lower to the chemically fertilized control (257.69 cm).

^{a, b, c}values with different superscript differ to P < 0.01.Growth curves correspond to the treatments: INICA-8 (discontinue lines), EcoMic^® (continous line) and EcoMic^®+INICA-8 (dotted line).Standard deviation: 15 DAS: 9.22, 45 DAS: 20.06, 75 DAS: 29.43, 105 DAS: 27.35, 135 DAS: 25.29, 165 DAS: 31.18.

Figure 4 Effect of microbial inoculants on the main stem length, at 15, 45, 75, 105, 135 and 165 DAS. 

The results obtained in this experiment show the superiority of the inoculated treatments with respect to AC in all the stages that were evaluated, where the combination of Glomus cubense with Azospirillum highlighted in the last three measurements (105 DAS, 135 DAS and 165 DAS). This superiority of the inoculants with respect to the control in these phases could be due to the synergistic effect of the Glomus cubense strain, which was applied in combination with the INICA-8 strain of Azospirillum brasilense. However, there was also a higher effect with respect to AC by each of the inoculants that were applied in a simple way in the first three measurements (15 DAS, 45 DAS and 75 DAS). However, these treatments did not exceed the fertilized controls.

The association of AMF with different mulberry genotypes can facilitate the adsorption of nitrogen and phosphorus by the plant (^{Ambika et al. 1994}). Likewise, the application of Azospirillum brasilense can reduce the requirements of nitrogen fertilizers, such as urea, which contributes to lower costs in mulberry (^{Das et al. 1994}).

^{Sánchez de la Cruz et al. (2008)} found that in greenhouse experiments the simple inoculation of G. intraradices, as well as A. brasilense, increased the plant height in wheat. However, the combination of these microorganisms did not exert a perceptible effect on the studied variables, despite there is a constant regulated humidity regime, which contradicts what was obtained in this experiment. It is possible that mechanisms specifically associated with the plant family, and not with the synergy of these microorganisms, influence on these divergent results. ^{Shaimaa and Massoud (2017)}, when inoculating a combination of Azotobacter and AMF in orange, observed a synergism that was reflected in the plants growth, in the extraction of N, P and K in leaves, and in the yield.

In this experiment, the superior response of plants to the application of chemical fertilizer coincides with that reported by ^{Baqual and Das (2006)}, who state that mulberry responds well to the application of nitrogen fertilizer as phosphoric.

Figure 5 shows the significantly higher effect of the treatment inoculated with EcoMic^® (1.70 cm) with respect to the rest of the treatments (P <0.001), although it shared common letters with the CFC (1.60 cm). In turn, the combination EcoMic^® + INICA-8 (1.50 cm) was higher to INICA-8 (1.33 cm) and the absolute control (1.23 cm), and shared common letters with the two fertilized controls.

^{a, b, c}Values with different superscript differ to P < 0.01Standard deviation: 0.21.

Figure 5 Effect of microbial inoculants on the main stem diameter 

The results obtained in this variable show a marked influence of the inoculation with AMF, in a simple way as in combination with Azospirillum. However, the treatment inoculated only with AMF higlighted, which coincides with that reported by ^{Pentón et al. (2011)} in an experiment with mulberry cuttings in a nursery.

Through the symbiosis that is established between AMF and plants, the absorption of some nutrients that are difficult to move in the soil (P, Fe, among others) is increased, due to the action of the external mycelium of AMF, linked to the plants root systems, and to the higher absorption of water, N, K and some micronutrients (^{Sanclemente et al. 2018}). This type of root branching also benefits the absorption of nutrients by increasing the root surface (^{Ruiz-Lozano 2016}). The AMFs improve the extraction of nutrients from the soil and their efficiency (^{Bitterlich and Franken 2016}), which brings with it greater exports of these.

^{Padma et al. (2000)}, when referring to the mycorrhizal symbiotic fungi that colonize the bark of mulberry roots, highlighted their important role in the efficiency of phosphorus mobilization and in the availability of a group of micronutrients, as well as in growth and plant development.

Several studies show that communities of diverse bacteria associated with AMF enhance mycorrhization and plant growth (^{Agnolucci et al. 2015}). This interaction between AMF-bacteria increases the colonization of both microorganisms in the host, because it increases the germination and growth of endophyte hyphae and increases the population of bacteria in the rhizosphere of plants in the presence of AMF (^{Fernández et al. 2016} and ^{Long et al. 2017}). However, the stimulation of plant growth in the presence of arbuscular mycorrhizal fungi does not necessarily have to be associated with the solubilization of elements that are not very mobile in the soil or at low concentrations. Mechanisms such as the production of phytohormones, vitamins or amino acids can be linked to the effects of these microorganisms (^{Siqueira 2010}).

Figure 6 shows the significant superiority (P <0.001) of the treatment inoculated with EcoMic^® + INICA-8 (2.96) with respect to the rest, especially with AC (1.70). The treatments inoculated in a simple way (EcoMic^® and INICA-8) shared common letters with the FCOM (2.42) and with the P₂O₅FC (2.25).

^{a, b, c}Values with different superscript differ to P < 0.01Transformed values are shown with √x on the Y axis, and original values within bars. Standard deviation: 0.57.

Figure 6 Effect of microbial inoculants on the number of branches 

The role of beneficial microorganisms in the development of agroproductive variables in mulberry is also evident in the number of branches. It is known that Azospirillum can also solubilize phosphates, which offers to the plant an important nutrient that has a positive effect on different physiological parameters (^{Tahir et al. 2013}). However, in this study, there was superiority in the results derived from the combination of the bacteria with respect to AMF. According to ^{Aguirre-Medina (2008)}, the simultaneous combination of growth-promoting rhizobacteria and arbuscular mycorrhizal fungi induces synergism, which is reflected in an increase in growth, phosphorus content in plants, and yield with respect to the plants separately inoculated. ^{Abd-Alla et al. (2014)} and ^{Bona et al. (2017)} consider that the result of the tripartite mutualistic symbiosis between AMF-bacteria-host is the increase in the development of the plant, since it increases its capacity to absorb more water and nutrients from the soil, such as nitrogen, phosphorus, potassium and microelements.

Various authors refer to results similar to that of this experiment, although with other beneficial microorganisms. ^{Nithya et al. (2011)} reported that in a study with mulberry, when inoculating the plants with a solubilizing phosphate bacteria and the beneficial fungus Aspergillus spp., the stem length was increased, as well as the number of branches and leaves. ^{Abdel-Rahman et al. (2011)} found that the effect of the co-inoculation of G. irradicans and Bacillus subtilis increased the height and number of branches of three varieties of basil, which surpassed the treatments where only each microorganism was inoculated.

Figure 7 highlights the significant superiority (P <0.001) of the EcoMic^® + INICA-8 (10.76) combined treatment with respect to the rest, with the exception of P₂O₅FC (10.28). The control fertilized with organic matter (9.22) was superior to INICA-8 (6.45), to AC (6.03) and EcoMic^® (5.90). These last three did not differ from each other.

^{a, b, c}Values with different superscript differ to P < 0.01Transformed values are shown with √x in the Y axis, and original values within barsStandard deviation: 2.19.

Figura 7 Effect of the microbial inoculants on the number of leaves 

In this study, the combined effect of Glomus cubense and Azospirillum brasilense was the inoculated treatment that had the most significant influence on the results of this variable, as it was statistically equal to P₂O₅FC. According to ^{Fernández and Rodríguez (2005)}, the mycorrhizosphere is the rhizosphere of a mycorrhizal plant, and it is in it where interactions with beneficial microorganisms with specific functions and interactions with pathogens take place. In many cases, the established interactions are positive, in which a synergism effect is recorded, where the AMF and the other microorganism produce growth increased, vigor and protection of the plant.

It is also known that the availability of P in soils is important for nitrogen absorption and its use in plants (^{Vafadar et al. 2014}). Therefore, more P available due to its solubilization by the inoculated AMF could lead to a better absorption of N or nitrogen fixation by Azospirillum, which would lead to an increase in the values of its variables as in the number of leaves. The co-inoculation of AMF-Azospirillum sp is an example of beneficial interaction, since the colonization of the roots by fungi stimulates the flow of carbohydrates from the canopy to the root, which can constitute sources of carbon for the bacteria growth (^{Costacurta 1995}).

Figure 8 shows that both EcoMic^® + INICA-8 (78.51g/plant) and FCOM (76.79 g/plant) were significantly higher to the rest of the treatments (P <0.001) and statistically similar to each other. The P₂O₅FC (64.97 g/plant) was higher to EcoMic^® (36.60 g/plant), the absolute control (30.10 g /plant) and INICA-8 (29.02 g/plant).

^{a, b, c}values with different superscript differ to P< 0.01Standard deviation: 24.82

Figure 8 Effect of microbial inoculants on the dry weight of leaves 

In this experiment, the DWL is the most important variable from the agronomic point of view, since it constitutes the basis of the mulberry forage production potential. ^{Sudhakar et al. (2000)} reported on the advantages of the application of bacterial biofertilizers in the production of leaves in this crop. ^{Baqual and Qayoom (2004)}, when applying Azotobacter and Azospirillum indistinctly to mulberry plants, observed an increase in the production of leaves and other variables. However, in this study , only the combination of Glomus cubense and A. brasilense led higher dry weight of leaves, with values similar to those of the application of organic fertilizer. Therefore, it is evident that the synergistic action of both microorganisms increased the number of leaves and other variables studied here.

The marked difference between the treatments in which the AMF and Azospirillum are combined with the control could be due to the amount of P absorbed by the plants due to the effect of the AMF applied. This hypothesis is supported by different authors who corroborate that mycorrhizae take up phosphates in the soil more efficiently through their hyphae that increase the volume of explored soil (^{Smith et al. 2011}). ^{Valerio (2016)} confirmed that the inoculation of corn with Claroideo glomus etunicatum leads to a significant increase in leaf area and dry weight of leaves.

^{Umakanth and Bagyaraj (1998)} observed that the double inoculation of S34 mulberry in nursery (suitable for dry areas) with Glomus fasciculatum and the diazotrophic bacterium Azotobacter chroococcum led to a considerable increase in plant growth and development. According to ^{Bellone and de Bellone (2012)}, the combination with Glomus and Azospirillum helps sugarcane to improve nitrogen availability and absorption in plants, which increases its biomass.

According to ^{Ram Rao et al. (2007)}, the combined inoculation of G. mosseae with A. chroococcum in mulberry showed its higher effect from the agronomic and economic point of view, with the improvement of soil fertility, quality of leaves, among other variables. ^{Vafadar et al. (2014)} reported that the dual inoculation of G. intraradices and A. chroococcum increased the dry weight of Stevia rebaudiana leaves. According to these authors, the ability of AMF to solubilize phosphorus in the roots ensures the bacteria a continuous fixation of atmospheric nitrogen, a process that could be one of the causes of the results obtained in this experiment.

The higher effect of organic fertilization on this variable is highlighted here. This result contradicts what was reported by ^{Bello et al. (2011)} about that the application of organic sources does not always benefit mulberry cultivation, especially when poor quality compost is used. This could show that the organic matter used in this experiment is of high quality.

Figure 9 shows that the IEI that predominated in this variable was the one obtained in the combined inoculation of EcoMic^® + INICA-8 (160.8 %), while the inoculation with EcoMic^® was lower, but with positive values (21.98 %). Also, the simple application of INICA-8 was not efficient.

Note that the EcoMic^®+INICA-8 combination was the one with the highest efficiency

Figure 9 Inoculation efficiency index based on DWL 

From the results of the inoculation efficiency calculation, a higher effect of the combination of the applied microorganisms can be seen. The simple application of Glomus cubense, and especially that of Azospirillum brasilense, did not exert a positive effect on the weight of leaves. This inefficiency of Azospirillum could be due to antagonistic factors that could affect the survival of the bacteria or its capacity as PGPR. These factors include the physical-chemical conditions of the soil, the genotype of the host and the ability of the bacteria to establish and compete with the native microflora (^{Pecina-Quintero et al. 2005}).

The interaction between AMF and bacteria increases the populations of both microorganisms in the rhizosphere of plants, which stimulates an increase in the volume of roots. This allows higher absorption of water and nutrients, in addition to increasing phosphorus solubilization, nitrogen fixation and resistance induction. In addition, tolerance to abiotic factors is improved, among others (^{Meng et al. 2015}). ^{Bécquer et al. (2019)} reported that the combined inoculation of G. cubense and Bradyrhizobium sp. in mulatto grass II, it resulted in an IEI of the aerial biomass higher than that obtained with the other forms of simple inoculation of these microorganisms.

It is concluded that the combined inoculation of EcoMic^® + INICA-8 exerted a higher effect on the studied variables. The variables that were most favored with the inoculation were the stem length and the number of branches. Simple inoculation of INICA-8 was inefficient in all variables.

It is recommended to evaluate the selected treatments in other field tests with Morus alba in different edaphoclimatic conditions and in various cuts, as well as to evaluate the inclusion of different levels of organic matter in the combinations of inoculants in Morus alba.

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Received: July 18, 2020; Accepted: October 10, 2020

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Conflict of interest: The author declare that there are no conflicts of interests among them

Author´s contribution: Carlos José Bécquer Granados: Original idea, desand conducting the experiment, carried out statistical analysis, manuscript writing. Adelaida Benita Puentes Pérez: Participated in the experiment. Arachely Cabrera: Participated in the experiment. María Hernández Obregón: Participated in the experiment. Ana Luisa Sánchez Reina: Participated in the experiment