Microorganisms used

Eleven isolates of Bacillus spp., from the collection of the Microbiology Laboratory of the National Technological Institute of Mexico-Technological Institute of Conkal were used. The bacteria were activated in nutrient agar (NA) for four to five days at 28 °C until autolysis and were preserved at 4 °C until use. The tests performed were the activity of ACC deaminase, production of indole acetic acid (IAA), production of siderophores and solubilization of calcium phosphate.

Activity of 1-aminocyclopropane 1-carboxylate deaminase (ACC deaminase)

The qualitative activity of the ACC deaminase of the 11 bacteria was performed by using the minimal medium Dworkin and Foster (DF) with ammonium sulfate (NH₄)2SO₄ (^{Penrose and Glick, 2003}). First, the strains were inoculated by stria in the DF medium, using (NH₄)₂SO₄ as a nitrogen source and after 48 h of growth, they were transferred to plates containing DF medium with 1-aminocyclopropane 1-carboxylate (ACC) (Sigma-Aldrich^®) as a nitrogen source, in the absence of ammonium sulfate. The petri dishes were then incubated for 3 days at 30 °C. Bacteria that showed growth were considered to have ACC deaminase activity.

Production of indole acetic acid (IAA)

To determine the microbial production of IAA, flasks containing 50 ml of nutritious broth supplemented with 1 g L^-1 of L-tryptophan were inoculated with each of the strains at a density of 1x10⁸ CFU ml^-1, subsequently, they were taken to an orbital agitator to be incubated for 72 h at 180 rpm and 30 °C. At the end, the supernatant was recovered by centrifugation at 3 000 by gravity (xg) for 15 min. To 1 ml of supernatant, 1 ml of Salkowski reagent was added. The mixture was left to stand at room temperature for 30 min and the concentration of IAA was determined in a spectrophotometer (Genesys 10UV) at 535 nm. To determine the concentration, a standard curve of 5 to 40 μg ml^-1 of IAA (Sigma) was used (^{Badía et al., 2011}; ^{Almoneafy et al., 2012}).

Siderophore production

To produce siderophores, the strains were previously grown in 7 ml of culture medium containing minimal salts (MS), left for 16 h in stirring at 120 rpm at 30 °C and after this time, 70 μl were taken from the bacterial culture which were transferred back to the MS medium, leaving in stirring for 24-30 h in the same conditions of stirring and temperature. Subsequently, the supernatant was recovered by centrifugation at 8 000 by gravity (xg) for 10 min, 1 ml of it was taken, mixing with 1 ml chrome azurol-S (CAS), the change in the coloration of the CAS reagent from blue to orange was considered positive for the production of siderophores (^{Alexander and Zuberer, 1991}).

Calcium phosphate solubilization

The qualitative activity of phosphate solubilization was performed by inoculating 8 μl of a bacterial suspension of 1x108 CFU ml^-1 in solid medium Pikovskaya (PKV) (^{Pradhan and Sukla, 2005}). Isolates that formed yellow halos in the culture medium containing bromophenol blue as an indicator were considered positive for phosphate solubilization, whose solubilization index (SI) was determined by the halo diameter (^{Qureshi et al., 2012}).

While the quantitative determination was made by inoculating by puncture in 25 ml of NBRIP medium with an initial pH of 7.2, which contained insoluble tricalcium phosphate as the only source of phosphorus (^{Mehta and Nautiyal, 2001}), the cultures were kept in constant stirring at 200 rpm for 5 days at 30 °C. At the end, the cultures were centrifuged at 8 000 by gravity (xg) for 10 min to recover the supernatant, in which soluble phosphorus was determined by the molybdenum blue method (^{Mussa et al., 2009}).

Bacillus spp., on the growth of seedlings of habanero pepper (C. chinense Jacq.)

The test was evaluated with the strains CBRF8, CBMT51 and CBMT2 of B. subtilis and BL18 of B. cereus, which showed better ability to solubilize phosphates, production of IAA, as well as ACC deaminase activity and selection was made based on a cluster analysis. For the test, seeds of habanero pepper var naranjo criollo were used, which were disinfected with 2% sodium hypochlorite, followed by three washes with sterile distilled water, then the seeds were sown in polystyrene trays with commercial sterile substrate Cosmopeat^®, giving the corresponding agronomic management (^{Soria et al., 2002}).

At 15 days after germination (DAG), an initial inoculation was performed at the stem level with a bacterial suspension adjusted to 1x10⁸ CFU ml^-1. A second application was made 28 days after germination (DAG). Subsequently, the transplant was performed in 32-ounce expanded polystyrene cups with Luvisol-type soil substrate with bovine manure in a 2:1 ratio, previously sterilized. At 60 DAG, height, stem diameter, number of leaves, leaf area, root volume, root length, fresh and dry biomass of the aerial part and root were measured.

A completely randomized experimental design was applied with four treatments (bacterial strains) with 10 repetitions and a control consisting of seedlings without bacterial inoculation. With the results obtained, an analysis of variance (Anova) was performed with a mean comparison test (Tukey, p≤ 0.05) with the help of the SAS statistical package version 9.3 for Windows (^{SAS Institute, 2010}).

Effect of Bacillus spp., on the yield of habanero pepper (C. chinense Jacq.)

To evaluate the effect of Bacillus strains on the production of habanero pepper, a test was carried out in an asymmetrical greenhouse located in the facilities of the Technological Institute of Conkal. Within which seedlings of 28 DAG were used, with two inoculations as described in the previous section. Subsequently, the transplant was performed in bags of 10 kg, which contained a mixture of Luvisol soil with bovine manure in a 2:1 ratio. A third inoculation was performed eight days after the transplant. Fertilization was carried out based on regional crop recommendations (^{Soria et al., 2002}).

The average maximum and minimum temperatures recorded inside the greenhouse were 35.4 and 24 °C, maintaining an average humidity of 73.4%. The plants were kept until 140 days, in which the same growth variables described above were evaluated, additionally, variables related to crop yield were included, such as: number of fruits per plant, polar and equatorial diameter of the fruit and total weight of fruits per plant. A completely randomized experimental design was applied with four treatments (bacterial strains) with eight repetitions and a control consisting of seedlings without bacterial inoculation. With the results obtained, an analysis of variance (Anova) was performed with a mean comparison test (Tukey, p≤ 0.05) with the help of the SAS statistical package version 9.3 for Windows (^{SAS Institute, 2010}).

Activity of 1-aminocyclopropane 1-carboxylate deaminase (ACC deaminase)

In the minimal medium DF, the growth of 10 of the 11 bacterial strains was observed, indicating that they have the ability to produce the enzyme ACC deaminase, with Bacillus sp., CBRF4 being the only one that did not show growth (Table 1). It is worth mentioning that differences in the growth of bacteria were observed, the isolates CBLMA4, CBCK44, CBCC58 and CBMT2 showed greater growth in the culture medium. This activity has been reported as part of the characteristics of microorganisms that have been used in the promotion of plant growth in pepper (C. annuum) and tomato (Lycopersicum esculentum) (^{Luna Martínez et al., 2013}; ^{Hernández-Forte et al., 2015}).

However, one of the most interesting applications of the strains that have this property is related to the reduction of environmental stress, where the application of Bacillus strains reduced the stress caused by soil salinity in corn plants (^{Misra and Singh, 2020}). On the other hand, biotic stress caused by diseases can also be decreased by ACC deaminase activity, where the use of a strain of Paenibacillus lentimorbus B-30488 (formerly Bacillus lentimorbus) reduces the stress caused by infection by the phytopathogenic fungus Sclerotium rolfsii in tomato (^{Dixit et al., 2016}).

Production of indole acetic acid (IAA) and siderophores

The 11 bacteria tested produced IAA in a range between 0.046 and 5.45 μg ml^-1. The strains CBMT2 and BL18 showed the highest production with 5.45 and 4.37 μg ml^-1, respectively (Table 1). ^{Luna-Martínez et al. (2013)}, in their research with strains of Bacillus spp., achieved the production of IAA in concentrations similar to those generated in this study, with which they obtained increases in the growth of seedlings of pepper (C. annuum L.) and tomato (S. lycopersicum L.), such as those generated in C. chinense Jacq. in this evaluation. The ability to produce IAA depends on several factors: the bacterial strain, the genes involved that moderate or regulate biosynthetic pathways and the presence of enzymes to convert IAA into its conjugated forms (^{Patten and Glick, 2002}).

Bacillus strains that produce IAA have been reported to promote growth in seedlings of soybean (G. max) (^{Wahyudi et al., 2011}), wheat (Triticum aestivum L.) (^{Abbasi et al., 2011}) and tomato (Solanum lycopersicum L.) (^{Almoneafy et al., 2012}). This hormone modulates cell growth and tissue differentiation, participates in the phenomena phototropism and gravitropism and has an important function during root and xylem formation (^{Vega-Celedón, 2016}). Studies in soil reveal that the microbial production of IAA depends on the presence of its precursor, tryptophan (^{Sarwar et al., 1992}). Regarding the production of siderophores, no coloration changes were observed in the CAS reagent, suggesting that under the conditions evaluated the bacteria did not synthesize siderophores (^{Alexander and Zuberer, 1991}).

Calcium phosphate solubilization

In in vitro tests, conducted in the laboratory, the 11 bacteria showed different solubilization indices (SI) of calcium phosphate, the greatest activity was observed at seven days of bacterial growth (^{Wahyudi et al., 2011}; ^{Almoneafy et al., 2012}). The SIs found were 2.1 to 2.76 mm, the highest SI was obtained with the strain CBRF4 (Table 1). ^{Qureshi et al. (2012)} reported SI values between 3.3 and 3.8, higher than those obtained in this study. However, the SIs reported in Bacillus spp., in this study agree with those reported by ^{Badía et al. (2011)}.

In the test in liquid medium, the study strains produced concentrations of 13.01 to 55.82 mg L^-1 (Table 1). The strain B. subtilis CBMT51 was the one that showed the highest solubilization with 55.82 mg L^-1, this result is similar to that reported for B. megaterium (MA06) with a solubilization of phosphorus of 56 mg L^-1 (^{Luna-Martínez et al., 2013}). It has been shown that the production of organic acids by bacteria lowers the pH, which in turn favors the solubilization of phosphates. In this study, a decrease in the final pH of the medium was found, fluctuating between 4.71 and 5.51 (Table 1), which partly explains that the solubilization of phosphates by bacteria is related to the decrease in pH (^{Mehta et al., 2010}; ^{Walpola and Yoon, 2013}).

Among the organic acids secreted during solubilization activity, lactic, isovaleric, isobutyric and acetic acid have been reported (Metha and Nautiyal, 2001). This activity is of great importance to improve the acquisition of phosphates by the plant, since due to its characteristics, when added in the form of fertilizer to the soil, phosphate is complexed with the calcium, iron or aluminum present, forming insoluble species, not available for the plant (^{Beltrán, 2014}). ^{Prakash and Kumar (2019)} report that the inoculation of Bacillus sp., STJP (previously characterized as a phosphate solubilizer) in Mentha arvensis increased the phosphorus content in roots, stems and leaves, also leading to an increase in biomass, and in the amount of essential oil.

Bacillus spp., on the growth of seedlings of habanero pepper (C. chinense Jacq.)

To perform the scrutiny of the strains with the best plant growth-promoting characteristics, a cluster analysis was carried out, selecting four strains: CBMT51, CBMT2, CBRF8 and BL18. Subsequently, these strains were inoculated in habanero pepper seedlings of 28 DAG. At 60 DAG, and it was found that the plants showed significant differences (p≤ 0.05) in height, number of leaves, leaf area, fresh weight and dry weight of the aerial part, root volume and fresh root weight. In the mean comparison test, the strain CBMT51 showed the best effect on plant growth (Table 2), compared to the other treatments and the control.

Only in the dry root weight (0.41 to 0.54 g) no differences between the treatments were observed. Several authors reported Bacillus spp., as a bacterium capable of promoting growth in C. annuum (^{Luna-Martínez et al., 2013}; ^{Amaresan et al., 2014}). ^{Ogugua et al. (2018)} report that the biomass of chili seedlings, after 35 days, increased 32.3%, similar to that reported in this study (34.6%). Gibberellins are other hormones also produced by strains of Bacillus, which have been involved in promoting the growth of chili seedlings, as reported by ^{Gil-Jae et al. (2004)}, which shows the versatility of the genus.

Effect of Bacillus spp., on the yield of habanero pepper (C. chinense Jacq.)

In the greenhouse test, it was observed that the strain B. subtilis CBMT51 presented the best effect by inducing a greater number of fruits and greater fresh weight of them, increasing by 79.5 and 58.8%, respectively, in relation to the control (Table 3). The strain B. subtilis CBMT2 induced increased height, stem diameter, number of shoots, total fresh and dry biomass (Table 3). Previous studies reported bacteria capable of promoting plant growth in C. annuum (^{Luna Martínez et al., 2013}; ^{Amaresan et al., 2014}). In habanero pepper inoculated with species of Azospirillum sp., increases in the aerial and root biomass of the crop were obtained (^{Canto-Martín et al., 2004}).

On the other hand, inoculations with species of B. amyloliquefaciens, B. licheniformes and B. subtilis in C. annuum increased height, number of fruits and yield (^{Datta et al., 2011}; ^{Yu et al., 2011}), changes that could be observed in this study with the application of B. subtilis CBMT51. ^{Peña-Yam et al. (2016)} report that the inoculation of seedlings of chili cv Jalapeño with the strain of Bacillus cereus ITC-BL18 increased the number of flower buds, which may also have occurred in the present work with B. subtilis CBMT51, which finally led to a greater number of fruits.

The improvement in the growth and development of plants when inoculated with this type of bacteria is mainly associated with the production of indole acetic acid and the solubilization of phosphates (^{López-Bucio et al., 2009}), properties that were demonstrated with the strains under study, with the strains B. subtilis CBMT51, B. cereus BL18 and B. subtilis CBMT2 being the ones that showed the best effect on habanero pepper plants for the variables evaluated. The use of Bacillus as a biofertilizer not only improves the development and production of plants, but recent studies also show that in chili fruits, it improves their organoleptic quality and increases the content of ascorbic acid and antioxidant activity (^{Cisternas-Jamet et al., 2020}).