Preparation and characteristics of liquid biofertilizer (bioslurry)

The bioslurry was elaborated according to the methodology for "enriched liquid biofertilizer" preparation, proposed by the ^{FAO (2013)}. To do this, a 220-liter plastic drum was used, where the components, listed in Table 1, were placed. Anaerobic digestion was performed for four months. The mixture was stirred weekly to homogenize the materials. At the end of the elaboration process, the mixture was filtered by a canvas fabric, and the liquid fraction was stored in a plastic drum with hermetic closure, protected from direct radiation. The final composition of the bioslurries in solution is presented in Table 2.

Table 1 Components incorporated into the bioslurry production process. 

Table 2 Physio-chemical characteristics of the dilutions used in the germination evaluation. 

Germination test

To evaluate the effect of bioslurry on germination, 25 tomato seeds were placed on filter paper in 90 mm Petri dishes, based on ^{Sobrero and Ronco (2004)}. The experimental design used was completely randomized, with six treatments, consisting in 4 cm³ per plate of bioslurry at 15% (Biol 15), 10% (Biol 10), 5% (Biol 5), 3% (Biol 3), 1% (Biol 1) and distilled water as control treatment (CW). Each treatment was repeated thrice. Once the seeds were placed on the plates, they were brought to the stove at 22 °C for 120 h in darkness.

The germination proportion (GP = number of seeds germinated in each dilution/number of seeds germinated in the control), length of hypocotyl, and radicle were determined. The effect of each dilution on the germination of tomato seeds was analyzed statistically using a non-parametric analysis of Kruskal Wallis (P<0.05), due to the lack of normality of data.

Seedling production test

One tomato seed per cell (17.15 cm³) was sown in plastic trays with 40 alveoli. The substrate used was Cocomix (Línea profesional, Carluccio, Bs. As., Argentina), composed of sphagnum brown peat, perlite, and coconut fiber. Irrigation and nutrition treatments were provided through immersion of the culture trays in plastic polypropylene containers, once a week. Periodically and according to demand by evapotranspiration, all containers were irrigated and remained in conditions close to field capacity during the test period. For irrigation and dilutions, free chlorine water, extracted from a subterranean well was used.

The treatments were: fertilization with a 5% solution of bioslurry (Biol 5); 10% solution of bioslurry (Biol 10); 15% solution of bioslurry (Biol 15); control with well-water (CW) and a fertilized control (CF), supplemented with nutrient complexes (Rootex 8-46-5.5, Cosmoflor S. A., Mexico, and Plant-Prod Iniciador, 10-52-10), Cosmoflor S. A., Canada, both dissolved at 2 g L^-1 in irrigation water. Prior to the incorporation, electrical conductivity expressed as dS m^-1 was measured in each treatment at 23 °C, obtaining CW: 1.070; Biol 5: 1.772; Biol 10: 2.372; Biol 15: 2.927 and CF: 2.280. The experimental design used was completely randomized with 15 experimental plots, where three replications were arranged per treatment. The effect of treatments on the emergence and development of seedlings was evaluated by analysis of variance and comparison of means (Fisher's LSD test; P<0.05), using the Infostat statistical software.

At 7, 10, 12, and 14 days after sowing (DAS), the number of emerged plants was recorded. With the data obtained, the percentage of emergence was calculated, as a relationship between the number of germinated seeds and the number of seeds sown (^{Prado-Urbina et al., 2015}). The evaluation of total fresh biomass (aerial and root) was carried out at 28 DAS, when seedlings of the CF treatment had three true leaves, and an approximate height of 10 cm from the base to the vegetative apex. From the central lines of each replica of the tray, ten seedlings were extracted, roots washed, and weighed.

Germination proportion

This variable was significantly (P<0.05) reduced by Biol 15 (0.20±0.12). Meanwhile, Biol 5 and Biol 10 (0.91±0.10 and 0.56±0.11) did not differ significantly from the Biol 15 and CW, although showing a tendency to affect germination. The values obtained with dilutions of Biol 1 and Biol 3 (0.98±0.03 and 1.00±0.00) presented no differences regarding to CW (1.00±0.00, Figure 1).

Figure 1 Germination proportion (number of seeds germinated in each dilution/number of seeds germinated in the control) of tomato seeds for each solution of bioslurry tested (Kruskal Wallis, P<0.05). Bars correspond to the standard deviation. CW: Water control; Biol 1: 1% bioslurry dilution; Biol 3: 3% dilution; Biol 5: 5% dilution; Biol 10: 10% dilution; and Biol 15: 15% dilution. 

Hypocotyl length

In this case, none of the dilutions differed significantly from the CW. However, at concentrations 1, 3 and 5% (4.17±0.54; 4.12±0.79 and 4.09±0.41 cm), hypocotyls were significantly longer than 15% (0.99±0.94 cm), showing values higher than CW (3.14±0.64 cm, Figure 2).

Figure 2 Length of tomato seedling hypocotyl for each bioslurry dilution. Averages with a common letter are not significantly different (Kruskal Wallis, P<0.05). Bars correspond to the standard deviation. CW: Water control; Biol 1: 1% bioslurry dilution; Biol 3: 3% dilution; Biol 5: 5% dilution; Biol 10: 10% dilution; and Biol 15: 15% dilution. 

Radicle length

Dilutions 1, 3, and 5% (4.01±0.34; 4.84±0.92 and 3.90±1.22 cm) did not differ from CW (5.42±1.51 cm), while 10 and 15% (3.00±0.47; 0.69±0.35 cm) were significantly lower than CW. In this sense, the responses generated by dilutions lesser than 5% indicated an absence of negative effects on tomato seedlings (Figure 3).

Figure 3 Tomato seedling radicle length for each bioslurry solution tested. Mean values with a common letter are not significantly different (P<0.05, Kruskal Wallis). Bars correspond to the standard deviation. CW: Water control; Biol 1: 1% bioslurry dilution; Biol 3: 3% dilution; Biol 5: 5% dilution; Biol 10: 10% dilution; and Biol 15: 15% dilution. 

In general, it was observed that after germination, tomato hypocotyl and radicle length decreased with increasing bioslurry concentration. According to ^{Medina et al. (2015)}, very high concentrations of bioslurries produced from sheep manure, inhibit the germination of lettuce seeds and limit the growth of the radicle, possibly by increasing the electrical conductivity of the solution. Likewise, ^{Goykovic et al. (2014)} verified in tomato seeds, the osmotic and non-ionic detrimental effects by saline solutions during the germination process.

^{Díaz (2017)} confirmed the presence of precursors of hormonal action, such as gibberellins, auxins, and cytokinins. In this sense, bioslurry concentrations between 2 and 5% presented a stimulating effect on lettuce, cotton, and alfalfa germination. Also, ^{Medina Vargas (1990)} points out that there are several hormonal precursors in the composition of bioslurry, but also certain repressors such as methionine.

It is important to consider that bioslurry is a complex solution of macro and micronutrients, microorganisms and growth precursors. Therefore, it became difficult to conclude and demonstrate that the seed's response is related to the presence of a particular chemical element, or a specific growth precursor. In this sense, the plants would react to the whole; that is, the interaction between chemical elements, growth precursors, and microbial population present in the bioslurry.

Emergence and biomass of seedlings

At 7 days, all treatments obtained significantly lower values than the control (53.5±7.37%). At 10 days, only the Biol 15 differed from the rest of the treatments with the lowest values. After 10 days, no statistical differences were observed among the treatments and the CW, although the trend observed at the beginning of the emergency assessment was maintained (Figure 4).

Figure 4 Emergence of tomato seedlings with solutions at different concentrations of bioslurry. Averages with a common letter are not significantly different (P<0.05, LSD Fisher). Bars correspond to the standard deviation. CW: Water control; CF: fertilized control; Biol 5: 5% bioslurry dilution; Biol 10: 10% dilution; and Biol 15: 15% dilution. 

The fresh biomass obtained in the seedlings after 28 days, showed a significantly higher response for fertilized control (CF), while CW presented lower values than the rest of the treatments. On the other hand, the solutions of Biol 5 and 10% (340.66±49.69; 357.75±67.26 mg) obtained a fresh weight lower than CF (707.79±108.34 mg) but significantly higher than Biol 15% (227.21±4.03 mg, Figure 5).

Figure 5 Fresh total biomass of tomato seedlings with solutions at different concentrations of bioslurry. Mean values with a common letter are not significantly different (P<0.05; LSD Fisher). Bars correspond to the standard deviation. CW: Water control; CF: Fertilized control; Biol 5: 5% bioslurry dilution; Biol 10: 10% dilution; and Biol 15: 15% dilution. 

Periodic applications of 15% bioslurry were less effective than 5 and 10% dilutions, both of which behaved similarly. These values correspond to the recommendations or the use of these types of biofertilizers (^{FAO, 2013}). Higher concentrations tend to limit the growth of seedlings.

Finally, fertilization plans to achieve complete seedling nutrition should be complemented by the design of a substrate containing organic compounds, as it was shown that both the development of the aerial part of the seedlings and the root system is favored (^{Moraes et al., 2021}).