Establishment and experimental design

The experiment was established under a completely randomized design (DCA) with a factorial arrangement of 2 × 2 × 2, where the study factors were: with or without inoculation with P. tolaasii (P), the sterilized substrate (SE) and substrate non-sterile (SNE) and the nutrient solution with Steiner (St) and vermicompost tea (Te).

A total of eight treatments with four repetitions each were evaluated. The experimental unit was a seedling tray with 25 serrano pepper seedlings, which were watered with sterilized water two to three times per day. Seedling emergence occurred after 12 days and they were thinned four days later, leaving one seedling per cavity.

Microbiological material

The P. tolaasii bacterial strain P61, belonging to the collection of the Soil Microbiology Laboratory of the Postgraduate Edaphology, was used as inoculum. The bacterium was inoculated six days after emergence, applying 1 mL of inoculum with a concentration of 1 × 10⁹ CFU mL^-1 at the base of each seedling.

Nutritive solution

On the third day after inoculation, seedling nutrition was started, using the Steiner 25% universal nutrient solution (^{Steiner, 1984}). As a second source of nutrition, vermicompost tea prepared according to the method of ^{González-Solano et al. (2014)}. Both solutions were adjusted to pH 5.5 and 24 days later their concentration was increased to 50% in both solutions until transplantation. The concentration was calculated based on the electrical conductivity of the Steiner solution.

Variables evaluated

56 days after planting, a destructive harvest was carried out. The variables evaluated were: stem diameter (mm), seedling height (cm), number of leaves, leaf area (cm²) with an LI-COR^® area measuring equipment, length (cm) and root volume (cm³) were determined with the water displacement method with a 25 mL cylinder.

In addition, NO₃ ^-, K⁺ and Ca²⁺ were quantified in petiole (sap) cell extract with Laquatwin^® ionometers. The seedlings were oven dried at 55 °C for 72 h and weighed to obtain the dry biomass of the aerial and root part. With the obtained values, the growth rates were calculated according to ^{Hunt (2017)} (Table 1).

Nutritional analysis

In the dry plant material, total nitrogen (N) was determined by the microKjeldahl method, phosphorus (P) and potassium (K) by means of wet digestion and plasma induction atomic emission spectroscopy using the methodology proposed by ^{Alcántar and Sandoval (1999)}.

Statistical analysis

The data obtained from the evaluated variables were submitted to the Shapiro-Wilk normality test (α= 0.05) and the Levene test for variance homogeneity. The variables of radical length, radical dry biomass, dry biomass of the aerial part, AFE, RAF, NO₃, K⁺, Ca²⁺ in cell extract and nutrient concentration (N, P, K), which met both tests, were applied analyzes of variance (Anova) and the Tukey mean comparison test (α= 0.05).

The variables height, stem diameter, number of leaves and leaf area that did not comply with the tests of homogeneity of variance or normality, were transformed to natural logarithm (ln) until normality and homoscedasticity were observed, later the Anova was calculated and the Tukey mean comparison (α= 0.05).

Data on root volume, dry leaf biomass did not meet both tests, so a Kruskal-Wallis non-parametric analysis and Wilcoxon’s rank summation were performed, according to Siegel (2015) methodology. For all cases, we worked with the statistical program Statistical Analysis System (SAS) Version 9.4 (^{SAS, 2014}).

Agronomic variables

The length and radical dry biomass, the dry biomass of the aerial part, AFE and RAF presented statistical differences between the treatments according to Tukey (p< 0.05) (Figure 1). Seedlings that developed in non-sterile substrate had a slight increase in root length, unlike those that grew in sterile substrate, however, in dry root biomass there are no differences between them.

Figure 1 a) radical length and radical dry biomass; b) dry biomass of the aerial part; c) specific leaf area; and d) ratio of leaf area of serrano pepper seedlings inoculated with P. tolaasii and different nutritional solution. Bars with different letters are statistically different according to Tukey’s mean test (p< 0.05). P= Pseudomonas tolaasii; S= substrate; E= sterilized; NE= Not sterilized; St= Steiner; Te= vermicompost tea. 

Regarding the nutrition source, when tea is applied as a solution, there is an increase in the length and dry biomass, although the Tukey test indicates in some cases that there are no differences (Figure 1a). It is interesting to see that the height and root biomass in seedlings without sterile substrate and with tea have the highest values of length and dry radical biomass.

Favorable responses to organic sources have been recorded by ^{David-Santoya et al. (2018)} for habanero pepper (Capsicum chinense Jacquin) and with inoculants to increase the radical length of C. annuum with Rhizobium etli, and with Trichoderma viride by ^{Vazallo et al. (2013)}. A greater root length in seedlings allows it to efficiently cope with transplantation and achieve rapid adaptation and absorption of water and nutrients (^{Vazallo et al., 2013}).

In the analysis of the aerial part, Figure 1b shows that the PSNESt and PSESt treatments registered the highest dry biomass (stem + leaves) of the seedlings, reaching 0.34 g and 0.33 g plant^-1, respectively. This means that in the aerial part of the seedling there is a response to the inoculation of Pseudomonas tolaasii and the application of Steiner nutrient solution regardless of the sterilization of the substrate.

This is attributed to the fact that P. tolaasii benefited from the absorption of available nutrients incorporated with Steiner solution (^{Angulo-Castro et al., 2018}) and to the improvement in root development when inoculated with rhizobacteria (^{Natarajan et al., 2012}). The inoculation of P. tolassi to strawberry plants favors the development of the plant (increase in leaf area and fresh weight of foliage), although it is not reflected in an increase in fruit production (^{Ortiz et al., 2016}).

The biomass of the aerial part of the seedlings grown in non-sterile substrate (Figure 1b) and watered with Steiner solution was very similar to those inoculated with Pseudomonas; however, the root length and biomass were reduced (Figure 1a) and this has a lot to do with the adaptation of seedlings to transplantation. In the AFE (Figure 1c) and RAF (Figure 1d) indices, the incorporation of Steiner solution without inoculation increased the values, in sterilized substrates.

Similar results have been recorded in pepper seedlings (^{Luna et al., 2013}) and in jalapeño peppers (^{Angulo-Castro et al., 2018}), when they were inoculated with Pseudomonas. ^{Amanullah et al., 2007}, indicate that high AFE values represent greater sheet thickness and a better balance between the potential capacity of photosynthesis and the cost of respiration.

These values were reached in seedlings where there was a sufficient supply of inorganic nutrients (St), which generates greater vigor of the plant, determined by the RAF, as well as a greater leaf thickness regulated by the AFE (^{Hunt, 2017}), index that allows explaining up to 80% of the variation in growth rates (^{Villar et al., 2004}).

In the Figure 2a shows the means of the variables of seedling height and stem diameter, where inoculation generated higher height (20% more) and stem diameter (between 13 and 5% more) between the PSNESt and PSESt treatments, regarding their controls (SNESt and SESt). Tukey's test shows highly significant differences between these two treatments and the others. Similar results were obtained by ^{Luna et al. (2013)}, with a significant increase in height and stem diameter of pepper seedlings when inoculating Bacillus subtilis up to 37%, compared to the control.

Figure 2 a) seedling height and stem diameter; and b) number of leaves and leaf area of inoculated serrano pepper seedlings and different nutrient solution. P= Pseudomonas tolaasii; S= substrate; E= sterilized; NE= not sterile; St= Steiner; Te= vermicompost tea. 

Indolacetic acid (AIA) produced by the inoculated strain is the main phytohormone that induces plant growth, by increasing cell division and tissue differentiation (^{Bhattacharyya and Jha, 2012}; ^{Glick, 2012}). In the case of poblano pepper, the increase in growth was 35% compared to the control due to the effect of inoculation with growth promoting bacteria, including P. tolassi (^{Quiroz-Sarmiento et al., 2019}).

In the number of leaves and leaf area per seedling, the treatment inoculated with P. tolaasii and watered with Steiner nutrient solution in sterile substrate (PSESt) was the one that registered the highest number of leaves, up to 12% more than the control (SESt), as shown in Figure 2b. In contrast, for the variable leaf area, the greatest leaf surface was observed (up to 20% more than the control), with the PSNESt treatment. The results of this variable indicate that the inoculation and the nutrient solution of Steiner at 25% supplies the balanced nutrients that are reflected in the increase in the number of leaves in all the seedlings that were watered with Steiner. Since rhizobacteria possibly provide growth benefits to plants when low amounts of chemical fertilizer are added to the soil that do not inhibit growth as established by ^{Bhattacharyya and Jha (2012)}; ^{Glick (2012)}.

Statistically significant differences in root volume were found according to the Kruskal-Wallis test, with the PSNETe treatment registering the highest volume with an average Wilcoxon range of 180.18 (data not shown), while the highest dry leaf biomass, was registered with the PSNESt treatment, with an average Wilcoxon range of 224.75 and the PSESt treatment with 216.72. Figure 3a shows the radical volume means, where it can be seen that the PSNETe treatment presented 3 cm³, surpassing 31% of its control (SNESt) and the SESt treatment obtained 2.2 cm³.

Figure 3 Box diagram contrasting: a) radical volume; and b) dry biomass of inoculated serrano pepper seedlings and different nutrient solution. P= Pseudomonas tolaasii; S= substrate; E= sterilized; NE= not sterile; St= Steiner; Te= vermicompost tea (Kruskal-Wallis non-parametric analysis). 

^{Pérez et al. (2015)} obtained increases in root volume of up to 64% in blackberry seedlings (Rubus glaucus L.) inoculated with Pseudomonas migulae compared to the treatment without inoculation. According to ^{Amara et al. (2015)}Pseudomonas is a genus of bacteria capable of producing and excreting auxins that are used by seedlings; the most investigated is indole-3-acetic acid, which is a phytohormone involved in stimulating radical growth (^{Glick, 2012}).

Figure 3b shows that the highest dry biomass was presented in the PSNESt treatments with an average of 0.253 g plant^-1 and PSESt up to 0.248 g plant^-1. ^{Parra-Terraza (2016)} identified that the supply of NO₃ ^- in the nutrient solution increased the dry biomass of leaves; however, with the participation of P. tolaasii this agronomic variable is 20% and 9% higher than its SNESt and SESt controls, respectively.

Nutritional variables

Statistical differences were found in the concentration of nitrates, potassium and calcium in the cellular petiole extract of the chili seedlings (p< 0.05. Figure 4a shows that the seedlings grown on non-sterile substrate, inoculated and watered with tea, they presented the lowest nitrate values (1 450 ppm) The seedlings grown with sterile substrate did not show significant statistical differences between them.

Figure 4 Nutritional variables: a) nitrates in sap; b) potassium in sap; c) calcium in sap; d) nitrogen; e) phosphorus; and f) potassium in inoculated serrano pepper seedlings and different nutrient solution. Columns with different letters are statistically different according to Tukey’s mean test (p< 0.05). P= Pseudomonas tolaasii; S= substrate; NE= not sterile; St= Steiner; Te= vermicompost tea. 

Potassium in the cell extract (Figure 4b) presented the highest values in seedlings grown on non-sterile substrate and only in the sterile control (SESt), whereas Ca²⁺, increased its content in seedlings with non-sterile substrate and the inoculation of P. tolassii regardless of the nutrient solution. Below these and statistically equal was the content in the non-sterile and sol-watered substrate plants, Steiner (Figure 4c).

The importance of knowing the chemical composition lies in the quality of the plant material of the seedlings, and the reserve of mineral nutrients, high concentrations of nitrogen and potassium, can influence the growth and survival of the seedlings (^{Villar et al., 2004}). ^{Shabayev (2012)} reported that inoculation with Pseudomonas, increases the absorption of nutrients by plants, growth, nutritional absorption and excretion of the root system associated with the secretion of phenolic compounds that has, among other things, the mission of promoting microniche of the rhizobacterium.

^{Moreno et al. (2018)} indicated that the RPCV participate in the gradual release of nutrients and contribute to the reserve of organic N and P from the soil, reducing the losses of N by leaching and the fixation of P (nutrient cycle). In addition, to supply micronutrients to crops (^{Glick, 2012}). In Figures 4d, e, f, the concentration of N, P and K in dry seedling biomass is shown, with highly significant differences between treatments (p< 0.05).

In the SESt treatment, it presented the highest concentration of N with an average of 55.5 g kg^-1; while the SNETe treatment registered the lowest concentration (33.6 g kg^-1). The other treatments were statistically similar. Regarding the concentration of P, the sterile substrate treatments with Steiner solution (PSESt and its control SESt) were better, which registered 4.15 and 4.13 g kg^-1 respectively.

Inoculation combined with Steiner’s nutrient solution (PSESt and PSNESt), increased the K concentration in dry biomass, surpassing the controls (SESt and SNESt) by 150% to 68%, on the other hand, the PSETe treatment registered the lowest concentration of K (6.56 g kg^-1), being 36% less than its control (SESt). ^{Jones (2014)} indicated that fertilization with Steiner solution improved seedling quality for transplanting, due to greater availability of nutrients.

While in the present investigation the supply of Steiner solution benefited the nutritive variables in seedlings and some agronomic variables. The proper balance and utilization of ions in the nutrient solution was achieved through the use of their universal nutrient solution formulas that improve the concentration of nutrients in plant matter (^{Steiner 1984}).

In the Table 3 summarizes the variables under study on the effect of inoculation with Pseudomonas tolaasii that when combined with Steiner nutrient solution in non-sterile substrates had better results in agronomic variables such as height, stem diameter, leaf area and dry aerial biomass, noting that the combination of inoculation with vermicompost tea in non-sterile substrate influences with increasing length and radical volume.

Not so when only vermicompost tea is applied, since it does not present any positive response, which means that the inoculation of P. tolaasii probably has positive effects when it interacts with other native or coinoculated microorganisms with different promoter capacity (substrate and tea) (^{Ahemad and Khan, 2012}; ^{Rojas et al., 2001}).

However, when only Steiner solution is applied, eliminating all the microorganisms, only the nutritional concentration benefits and influences the growth rates because the nutrition applied to the plants can affect the structure of the bacterial communities in the rhizosphere (^{Glick, 2012}; ^{Shabayev, 2012}). And the colonization process is a decisive step in the interaction between bacteria and plants, the latter secreting nutrient-rich exudates that are used by bacteria in the first stages of recognition (^{Abaid-Ullah et al., 2015}).

The hypothesis that is presented in the present study is that the inoculation of P. tolaasii combined with vermicompost tea favors the agronomic and nutritional development of serrano pepper seedlings. Therefore, the objective of the present investigation was to quantify the effect of the inoculation of Pseudomonas tolaasii on the physiology and development of serrano pepper (Capsicum annuum L.) seedlings using different nutritional solutions.