Experiment location

The work was developed in the productive sector of the Zona Olmeca Campus belonging to the National Technological Institute of Mexico located in Villa Ocuiltzapotlán, Centro, Tabasco, at coordinates 18 07’ 7.75” and 18º 09’ 39” north latitude and 92º 52’ 24” and 92º 50’ 55” west longitude. The study area presents a warm humid climate with abundant rainfall in summer, average annual temperature of 27 ºC and precipitation of 1 926 mm per year (^{INEGI, 2018}).

Crop management

Cuttings of Stevia rebaudiana variety Morita II rooted in commercial peat for 30 days were used, the cuttings were obtained in Tekax, Yucatán, Mexico. The variety Morita II is a Japanese variety, has higher dry leaf yields and product quality than other varieties including the landrace variety (^{Herrera et al., 2012}), also has a lower content of steviosides that cause the bitter taste of the leaf and has a higher amount of rebaudioside A, a product demanded by the industry (^{Ramírez et al., 2011}).

The crop was established in the autumn-winter period (August 2020), in cultivation beds with the following dimensions: 0.7 m wide, 25 cm long and 0.3 m high. The soil used was in alluvial plain (^{Palma-López et al., 2007}), the soil composition is shown in Table 1. The cultivation beds were covered with silver plastic mulch.

The irrigation was by drip, an irrigation of 0.5 L day^-1 was provided at the time of transplantation and supplemental irrigation was eventually given according to the requirement of the crop, since most of the water requirements of the plant were covered with precipitation. The sowing was carried out with a staggered arrangement with a distance of 30 cm between plants and 20 cm between rows, and at the time of transplantation a tipping was carried out to homogenize the plants.

Treatments

The treatments used were: bovine manure (BM), sheep manure (SM), vermicompost (VC) and chemical fertilizer (CF: control) with urea: 46-0-0, the nutrient analysis of the treatments is reported on a dry matter basis (Table 2). The manure was collected in the corrals of the region and exposed to the sun to standardize the moisture, the vermicompost was obtained from the microenterprise ‘Rancho Alegre’ of the municipality of Comalcalco, Tabasco.

Soil analysis was considered to calculate the dose of organic amendments. Table 1, and the amount of manure used from the dose of 180 kg ha^-1 of N (^{Casaccia and Álvarez, 2006}) was determined by the equation:

Where: DF= dose of fertilizer; ND= nutrient demand; NSS= nutrient supply by soil; % NC= percentage of nutrient in the compost; with which the following was obtained: BM= 857 g m^-2, SM= 392 g m^-2 and VC= 1 280 g m^-2.

Organic amendments were applied 100% at the time of transplantation, while for chemical fertilization 50% was applied at transplantation and the remaining 50% 30 days after transplantation (dat). Seven days after planting, a formation pruning was carried out by removing the apical bud and 20 dat the formation pruning was carried out.

Growth variables

The percentage of survival at transplantation was determined four days after transplantation, in which the edge effect was considered. The growth variables evaluated were plant height (cm), number of shoots at 30 and 45 dat. At 60 dat, root length (cm) was measured, and dry biomass of leaves, stems and roots was weighed on a digital scale (Ohaus, Pioneer) in grams. For the determination of dry biomass, leaves, stems and roots of plants previously washed with distilled water were separated, dried at 60 ºC in a forced-air oven (Lumistell, HTP-42 series) until constant weight.

Foliar nutrient concentration

The sampling for the foliar chemical analysis was performed at 60 dat. TN was determined by the modified micro-Kjeldahl method to include nitrates, P by colorimetry with molybdate and ammonium vanadate, K and Na by atomic emission and Mg, Na, Fe, Mn, Cu and Zn by atomic absorption with a Perkin Elmer Analyst 100^® atomic absorption equipment.

Experimental design and statistical analysis

The experimental design was completely randomized, with three repetitions, four plants were used as an experimental unit. The data in percentages were normalized with the square root of the arcsine and the data obtained were analyzed using an Anova, the variables with significance were subjected to a comparison of means with the Tukey test (p≤ 0.05) using the statistical program Statical Analysis Software version 9.1 (^{SAS, 2003}).

Survival percentage

The percentage of survival of stevia was greater than 81% in all treatments; however, vermicompost (VC) presented the maximum survival with 98.9%, while sheep manure (SM) obtained the lowest percentage with 81.5% (Figure 1), which means that the treatments presented significant differences (p≤ 0.05), while the chemical fertilizer treatment (CF; control) was statistically equal to the treatments VC and BM (Figure 1).

Figure 1 Percentage of survival at transplantation of Stevia rebaudiana Bertoni var. Morita II, established with different organic amendments. EB= bovine manure (BM), EO= sheep manure (SM); LC= vermicompost (VC); FQ= chemical fertilizer (CF). Means with equal letters are not statistically different (Tukey, p≤ 0.05). 

The differences in the percentage of survival of plants established with vermicompost were favored because VC reduces the C:N ratio, the phytotoxicity of organic residues and has low electrical conductivity; this can be observed with the chemical analysis of each treatment (Table 2); likewise, ^{Guerrero et al. (2012}) mention that VC is a stable product that avoids risks in the soil-plant system associated with soil oxidation.

On the other hand, sheep manure used without prior decomposition process possibly affected the survival of plants by raising the microbial activity of the soil and facilitating the splitting of nitrogenous organic substances that generate ammoniacal nitrogen, which can cause root burning. It is important to mention that weather conditions could affect the survival of plants, because rainfall was higher than 300 mm per month and temperatures above 26 ºC (^{CONAGUA, 2020}), environmental factors that contribute to the development and growth of stevia.

Plant height

Differences (p≤ 0.05) in plant height were found between treatments at 30, 45 and 60 dat (Figure 2). The plants grown with VC presented the highest growth in the three dates evaluated with 14.75, 23.767 and 40 cm respectively, and it exceeded the control (CF) by 34.1, 57.8 and 116.2%, which presented a height of 11, 15 and 18.5 cm, respectively.

Figure 2 Plant height of Stevia rebaudiana Bertoni var. Morita with different organic amendments, at 30, 45 and 60 days after transplantation. EB= bovine manure (BM); EO= sheep manure (SM); LC= vermicompost (VC); FQ= chemical fertilizer (CF). Means with equal letters are not statistically different (Tukey, p≤ 0.05). 

Our height results were lower than those reported by ^{González et al. (2019}); ^{Karimi et al. (2019}); ^{Sheikhalipour et al. (2021}) in other cultivation systems. However, the results of this experiment coincide with what was reported by ^{Aghighi et al. (2019}) and with ^{Romero et al. (2017}), who mention that at a higher proportion of vermicompost in plants of Stevia pilosa Lag. and Stevia tomentosa H. B. K., plant growth is improved by increasing nutrient availability.

In this regard, ^{Domínguez et al. (2010}) mention that the increase in the growth and productivity of horticultural crops fertilized with vermicompost is due to the fact that this material improves the physical and chemical structure of the soil and favors other biological mechanisms that stimulate plant growth, such as the production of humic acids, free enzymes and plant growth-regulating substances.

Number of shoots

In number of shoots there were no differences (p≤ 0.05) between treatments at 30 dat, all treatments produced on average 5.1 shoots per plant (Table 3). At 45 days of cultivation, the VC treatment presented the highest number of shoots per plant (23.1), surpassing the rest of the treatments. These results are similar to those reported by ^{Villalba et al. (2018}), who point out that vermicompost creates better conditions for the growth of stevia, producing three times more shoots than chicken manure; on the other hand, ^{Avilés et al. (2019}) mention that Leucaena leucocephala plants grown with vermicompost presented 18.3% more shoots than the control (without vermicompost). In addition, ^{González et al. (2019}) found a higher number of branches (33.8) per plant, at the age of 60 days in greenhouse conditions, grown in black soil with vermicompost.

Root length

The use of vermicompost favored the growth of the roots of the plant (60 dat) as they presented the longest length with 17.7 cm, compared to the treatments BM, SM and CF (control) with a shorter length of 15, 14.6 and 14.5 cm respectively (Table 3). In relation to this, ^{Salazar et al. (2018}) report that vermicompost modifies the physical, chemical and microbiological properties of the soil, which positively affects the agronomic variables of the Stevia rebaudiana crop, these results coincide with ^{Liu et al. (2011}), who report that, from 60 dat, there is a greater vigor of Stevia rebaudiana roots in plants grown with organic residues compared to chemically fertilized plants, ^{Campos et al. (2020}) also report that the use of organic amendments to the soil favors root growth of plants, by improving soil structure, increasing microbial activity, nutrient absorption and moisture retention.

On the other hand, ^{Gayosso et al. (2021}) mention that the roots of the plants in containers are distributed according to the physical characteristics of the substrate, such as porosity, and consequently the distribution of water. It is possible that the use of vermicompost as an organic amendment has improved the physical properties, as well as the water availability and biological activity of the soil (^{Cauich et al., 2018}; ^{Salazar et al., 2018}), which was reflected in an increase in the root system.

Dry biomass

At 60 dat, the production of dry leaf biomass in treatments with organic amendments showed no significant differences (p≤ 0.05) between them, compared to the control (Table 4). Nevertheless, the total dry biomass of plants with vermicompost, bovine manure and sheep manure were superior to the biomass of plants grown with chemical fertilizers (CF). The production of dry biomass with organic amendments coincides with the results obtained by ^{Liu et al. (2011}) at 80 dat, where the dry biomass per plant in stevia was higher with organic fertilizers than with chemical fertilizers; on the other hand, ^{Velázquez et al. (2017}) mention that the application of bovine manure in stevia was significantly higher than the control in production of dry leaf biomass per plant.

Foliar nutrient concentration

For N, P, K, Ca, Mg, Na and Mn, no significant differences (p≤ 0.05) were observed in foliar nutrient concentration analyzed at 60 days after transplantation (Table 5). With respect to N, it is due to the fact that the same amount was applied in all treatments; however, there were statistical differences in the content of Cu, Fe and Zn (Table 2); nevertheless, these results were lower than those obtained by ^{Yu-Ming et al. (2021}), the differences may be due to the experiment being conducted under other growing conditions.

The concentrations of macronutrients in dry biomass in some cases are not related to the amounts present in organic amendments. For example, for Cu in the treatments BM and SM, there were no significant differences between them, despite the fact that SM contains 28.5% more Cu than BM. With respect to Fe, bovine manure contains the highest concentration, and in the nutrient analysis of the plant, VC reflected the highest concentrations. For Zn, sheep manure showed the highest concentration; however, in the nutrient analysis there were no statistically significant differences with the other fertilizers, although CF was statistically different, with less of this element.