Introduction

The production of vegetables is a very important activity for the global and national agricultural sector, among the horticultural crops of economic and nutritional interest for its fresh or processed consumption. The red pepper (Capsicum annuum L.) is in seventh place worldwide (^{FAO, 2015}, ^{Acevedo-Chávez and Sánchez-Chávez, 2017}). In Mexico, the production of red pepper has shown an increase due mainly to its export to the United States and Canada, which generates a significant amount of foreign currency, nationally the cultivated area of this vegetable is 2 641.43 ha with a yield average of 42.08 t ha^-1, which represents an economic value of 519 321.21 million pesos (^{SIAP-SAGARPA, 2015}, ^{Acevedo-Chávez and Sánchez-Chávez, 2017}).

Due to the implementation of intensive agricultural production schemes, such as that used in the cultivation of red pepper to obtain greater volumes of fruit, the constant and excessive use of chemical fertilizers has been promoted, which besides being costly are highly polluting for soil, water and human health (^{Sánchez et al., 2012}; ^{Schulz and Glaser, 2012}).

Faced with this scenario of intensive agriculture based on chemical and expensive nutritional inputs, the growing use of growth promoting rhizobacteria in plants (PGPR), is promoted by its ability to directly or indirectly induce root health and growth and foliage of plants through the production of growth regulators such as auxins, gibberellins and cytosines (^{Lugtenberg and Kamilova, 2009}; ^{Belimov et al., 2015}), fixation of atmospheric N₂, solubilization of insoluble phosphorus (^{Naz and Bano, 2010}; ^{Zaidi et al., 2015}; ^{Pankievicz et al., 2015}), synthesis of siderophores, antibiotics, antifungal metabolites, salicylic acid, cyanide (^{Adhikari et al., 2013}; ^{Sunar et al., 2013}; ^{Kamou et al., 2015}) and induction of resistance in plants (^{Bakker et al., 2013}; ^{Vacheron et al., 2013}). Among the diversity of PGPR are a large number of species grouped in the genera Pseudomonas, Azospirillum, Burkholderia, Serratia, Azotobacter, Enterobacter, Streptomyces, Bacillus, Rhizobium, among others (^{Berg, 2009}, ^{Adhikari et al., 2013}, ^{Kamou et al., 2015}).

The form of inoculation of rhizobacteria on plants is versatile and simple, since they can be applied to the seed, root or soil (^{Shen et al., 2013}; ^{Cordero-Ramírez et al., 2013}; ^{Zaidi et al., 2015}). The response of plants to inoculation varies considerably and is a function of the species of rhizobacteria, the host, soil type, environmental conditions, inoculum concentration and mode of inoculation (^{Becerra-Castro et al., 2011}; Conejo et al., 2014). The methods of inoculation of the PGPR are determinant in the process of colonization, activity and permanence of the bacterial cells in the rhizosphere of the plants, besides indirectly influencing the mechanisms of action that promote growth in plants (^{Krzyzanowska et al., 2012}; ^{He et al., 2016}).

Among the methods of inoculation of the rhizobacteria, the microcapsules stand out, which have proven to be more efficient than the liquid form, due to their quality of providing protection and stability to the bacterial cells, allowing them to survive for longer in the rhizosphere of the cells. plants (^{Bashan et al., 2014}; ^{Schoebitz Belchí, 2016}). In this regard, ^{Sivakumar et al. (2014)} when evaluating the effect of Bacillus megaterium microcapsules supplemented with humic acids in rice plants, report outstanding results in the growth of plants, highlighting the action of humic acids in microcapsules by providing chemical stability and availability of C and N, generating a greater number of bacterial cells.

While ^{Abo-Kora et al. (2016)} when inoculating tomato plants with microcapsules and bacterial suspension of Pseudomonas fluorescens, Azotobacter chroorcoccum, Bacillus polymyxa and Azospirillum brasilense, found that both forms of inoculation generated favorable effects on the growth of plants, with the standing out microcapsules of P. fluorescens and A. Brazilian in plant height and root length, respectively. While ^{Schoebitz et al. (2013)} when evaluating in wheat plants inoculated with microcapsules based on sodium alginate and a combination of potato starch with sodium alginate, containing in both formulations the rhizobacterial strains P. fluorescens and Serratia sp. report that the rhizobacteria in both presentations promoted plant height, dry biomass and P foliar content, so they point out that the microcapsule method improves the effect of rhizobacteria by acting as mini-reactors that give bacterial cells stabilization, protection, population increase and progressive release around the rhizosphere of the plants where they are applied (^{Malusa et al., 2012}; ^{Schoebitz et al., 2013}).

Therefore, knowing the effects of different methods of inoculation of rhizobacterial species, can help in the development of efficient biofertilization programs that facilitate rhizobacterial cells expression of their qualities as growth promoters and productivity in plants. Therefore, the objective of the present work was to evaluate the effect of the inoculation of microcapsules and liquid culture of three strains of Pseudomonas putida on the growth and yield of red pepper in greenhouse.

Materials and methods

Results and discussion

The obtained results indicate that the red pepper plants inoculated with the microcapsules and liquid culture of the three rhizobacterial strains individually and combined showed significant differences (p˂ 0.05) in the morphological variables of the plant, yield and content of total soluble solids of fruit (Table 1). The strain FA-56_mc applied to the red pepper plants by means of microcapsules of alginate significantly increased (p˂ 0.05) the height, root volume, dry biomass of the plant and fruit yield in 31.50, 32.56, 71.06 and 45.10% respectively, in comparison with the control plants. The plants inoculated with the strain FA-60mc and the combination of the three strains (MIX_mc) applied in microcapsules obtained significant increases (p˂ 0.05) for root length and stem diameter of 32.56 and 29.89% respectively, compared with plants without inoculation.

In contrast, for the fresh plant biomass variable, the treatment that showed the best response with respect to the control was the strain FA-60_cl in liquid form, obtaining a significant increase of 30.2% (p˂ 0.05). Regarding the percentage content of total soluble solids (°Brix) in mature fruits of red pepper, there are significant differences (P˂ 0.05) between treatments, highlighting the strain FA-56 applied by microcapsules of alginate and liquid culture with increments of 12.5% in both cases in relation to the fruits of control plants.

The use of rhizobacterial strains of different genera such as Pseudomonas, Bacillus, Azospirillum, Serratia, among others, have been widely studied, particularly for their quality to promote vegetative growth and productivity in red pepper crops (^{Gupta et al., 2015}), tomato (^{Agrawal and Agrawal, 2013}), rice (^{García et al., 2010}), soybeans (^{Husen et al., 2011}), maize (^{Hungria et al., 2010}) and potatoes (^{Arseneault et al., 2015}).

This stimulating activity is related to the ability of rhizobacteria to synthesize plant growth regulators such as auxins, gibberellins, cytokinins and vitamins, antagonistic metabolites such as siderophores and hydrocyanic acid, as well as for their ability to facilitate the assimilation of nutrients through the solubilization of phosphorus, biological fixation of atmospheric nitrogen and ion chelation (^{Aguado-Santacruz et al., 2012}; ^{Vacheron et al., 2013}; ^{Ahemad and Kibret, 2014}; ^{Bashan et al., 2014}).

Regarding ^{Sivakumar et al. (2014)} when evaluating the effect of Bacillus megaterium by microcapsules of alginate supplemented with humic acids in rice plants, indicate having obtained differences in seed germination, root length and stem height in relation to the control plants, also mention that the growth of rice plants was stimulated by the metabolic activity of the B. megaterium strain; however, the addition of humic acids in the microcapsules provided chemical stability and an organic nutritional source of C and N, which maintained a higher number of live bacterial cells. For ^{Ahirwar et al. (2015)} the inoculation of the SS5 strain of Pseudomonas fluorescens in tomato plants showed significant increases in root length, plant height and fruit yield, indicating that the plants inoculated with the SS5 strain in pot and field surpassed the control plants in 19 and 57% yield, respectively.

On the other hand, ^{Abo-Kora et al. (2016)} inoculated tomato plants with microcapsules of alginate and bacterial suspension based on Pseudomonas fluorescens, Azotobacter chroorcoccum, Bacillus polymyxa and Azospirillum brasilense, finding favorable effects on the growth of plants with both forms of inoculation of the rhizobacterial strains, excelling the microcapsules of P. fluorescens and A. brasilense. In this regard ^{Schoebitz et al. (2013)} when inoculating wheat plants with microcapsules of sodium alginate and potato starch combined with sodium alginate, both combinations with strains of Pseudomonas fluorescens and Serratia sp., Found that these significantly promote plant height, dry biomass and foliar content of P, attributing that encapsulates improve the effect of rhizobacteria by acting as mini-reactors that give bacterial cells stabilization, protection, population increase and progressive release around the rhizosphere of the plants where they are applied.

In lettuce plants ^{Rekha et al. (2007)} applied microcapsules of sodium alginate and liquid suspension containing the rhizobacteria of the species P. putida CC-FR2-4 and B. subtilis CC-pg104, in both forms of inoculation the plants treated with the strains showed significant increases in length radical and stem height in relation to the control plants, conclude that the inoculation of the lettuce plants with P. putida and B. subtilis by means of alginate encapsulates vegetative growth is promoted in a similar way to the liquid suspension, so which the technique of encapsulation of rhizobacteria could be feasible for its use in the agricultural industry of biofertilizers.

Regarding the content of soluble total solids °Brix, some reports attribute their increase in fruits from biofertilized plants due to the capacity of the rhizobacteria to facilitate the assimilation of essential nutrients and stimulation by means of growth regulators, such metabolic activities propitiated directly by the action of the rhizobacteria induce the production of ethylene (^{Gamalero and Glick, 2015}), which promotes the synthesis of sugar-reducing enzymes present in the cell wall of the fruit, generating simple sugars that increase the concentration of total soluble solids (°Brix) in the fruits during the maturation process (^{Ordookhani and Zare, 2011}; ^{Vázquez-Ovando et al., 2012}).

The presence of rhizobacterial populations (UFC) quantified in roots of red pepper plants, inoculated with microcapsules of alginate and liquid culture with strains of P. putida showed significant differences (p˂ 0.05) between treatments (Figure 1). For microcapsules, the highest population of UFC (10⁸ g^-1 of fresh root tissue) was found in the three strains individually and in combination, highlighting the population obtained from strain FA-56 with 590 UFC (10⁸ g^-1). This same strain also stood out in the condition of liquid bacterial culture when obtaining the highest population number with 492 UFC (10⁸ g^-1). Research conducted on the use of rhizobacteria, indicate that the population rate of bacterial cells present in the rhizosphere of plants depends on the organic compounds produced by the radical exudates of these, among which are amino acids, organic acids, phenols, phytohormones (auxins, gibberellins and cytokinins), sugars, vitamins and enzymes (^{Berg, 2009}; ^{Adhikari et al., 2013}; ^{Bashan et al., 2014}).

Figure 1 Colony forming units (UFC) quantified in root of red pepper plants var. California Wonder, inoculated with alginate microcapsules and liquid culture based on P. putida strains FA-8, FA-56, FA-60, MIX (mixture of the three strains) and control (plants without rhizobacteria only inorganic fertilization) at 100 days after the transplant in the greenhouse. Different letters on the bars show significant differences according to Duncan’s multiple range test (p˂ 0.05). 

The quantity and quality of the radical exudates promotes in the rhizobacteria the competition for these metabolites, as well as for the place they occupy on the root of the plant, being the junctions between the epidermal cells and the area where the roots emerge with greater attraction, adhesion, activity and microbial population (^{Lugtenberg and Kamilova, 2009}, ^{Vacheron et al., 2013}).

Conclusions

The response of the red pepper plants inoculated with the FA-56 strain in the form of microcapsules showed the greatest increases in height, root volume, dry biomass, yield and content of soluble fruit solids. The same strain encapsulated and in liquid suspension had the largest rhizobacterial population in root (UFC).

The UFC populations determined in the microcapsule treatments indicate the possibility that the application of P. putida rhizobacteria by means of the microencapsulation technique facilitates the gradual release of the cells, improving their adhesion and colonization in the roots, inducing better effect as promoter agents of plant growth, which can be a viable alternative as biofertilizer for the cultivation of red pepper and its sustainable production.

More studies are needed to corroborate the potential of the strains of P. putida microencapsulated for their use as agents that stimulate the growth and productivity of red pepper plants.