Low rate and high variability in germination capacity are considered the main agronomic limitations for the domestication of chiltepin, for this reason, rhizobacteria application to improve the germination process is a valuable technology20. In this study, Bacillus spp. improved the germination parameters of chiltepin. Several authors have reported that Bacillus spp. promotes the germination of Capsicum genus species. Bolaños et al. 30 reported a 23.61% higher germination in chili pepper seeds (C. annuum L.) inoculated with B. licheniformis M2-7. In another study, B. subtilis increased the germination of "Kandil Dolma" peppers by 14.68% 31. The increases can be attributed to the production of bacterial-origin growth regulators, such as gibberellins, cytokinins, and auxins, as there is a direct relationship between the concentration of phytohormones and the physiological state of the chiltepin seed 32. According to Chowdhury et al. 33, the production of growth regulators is the main mechanism of Bacillus sp. LBF-01 is involved in the germination of chili seeds. For instance, gibberellins induce nutrient mobilization and the hydrolases synthesis that act in the degradation of storage products that constitute the endosperm34. Similarly, the production of exogenous auxins31, allows early root development32, through cell division and wall extensibility by enzymatic action35. In this study, chiltepin seeds had an overall germination time of 7.3 days, compared to the MGT reported in C. chinense Jacq. (6.72 days) and C. frutescens L. (8.8 days)36, 37. However, treatment with Bacillus spp. isolates reduced the germination time to as low as 0.46 days. This is consistent with the results reported by Sosa et al. 36, who showed a reduction of 0.66 days in germination of habanero pepper seeds inoculated with Bacillus sp. CBCRF12. Additionally, the treatments accelerated the germination process in chiltepin with up to 0.279 seeds germinated/day. This result is higher than what is reported in the literature (0.017 seeds/day)36. Furthermore, the vigor, germination, and slenderness indices were used as indicators of seed potency. Reed et al. 38 mention that the vigor index combines properties to determine the potential performance of viable seeds during the germination process. For its part, the germination index is an indicator that allows the integration of the germination percentage and root growth39. Bacillus spp. isolates improved the vigor and germination indices. In comparison, Jayapala et al. 40 observed an 8.35 and 9.59% increase in the vigor and germination index, respectively, in chili plants through biopriming with carboxymethylcellulose as an adhesive agent to Bacillus sp. BSp.3/aM. Otherwise, the slenderness index, calculated as the ratio between height and root collar diameter, was used to indicate seedling quality and resistance41. Values below 6.0 correspond to good-quality plants42. In this study, 16% of the treatments had values below the index, indicating more robust seedlings. These results suggest that treatment with Bacillus genus isolates favors the successful establishment of seedlings by accelerating root development, enabling the search for soil moisture, and rapid shoot growth to reach the surface. The acceleration of the germination process is of particular interest under stress conditions38.
Table 7
RWC and proline in C. annuum var. glabrisculum leaves subjected to drought stress. Data are represented by the average of 6 and 3 repetitions, respectively. Different letters indicate significant statistical differences according to the Scott Knott test (p ≤ 0.05).
| Treatment | Prolina (µM / g de hoja) | RWC (%) |
| C abs | 4.44 ± 0.70 c | 100.04 ± 13.30 a |
| C Xp Amino® | 136.60 ± 32.7 a | 46.76 ± 10.18 b |
| C - | 135.21 ± 1.09 a | 28.56 ± 09.43 c |
| Bc24-4 | 63.03 ± 14.6 b | 50.49 ± 12.45 b |
| Bc25-7 | 84.77 ± 14.1 b | 57.71 ± 21.54 b |
| Bc30-2 | 53.55 ± 13.3 b | 45.64 ± 07.58 b |
Under greenhouse conditions, seedlings treated with rhizobacteria increased root length, stem diameter, leaf fresh weight, and chlorophyll a. Other authors have reported positive effects on the growth of chili plants inoculated in the vegetative stage. García et al. 29 and Kazerooni et al. 27 observed a 33.3% increase in root length in chiltepin and habanero chili plants treated with B. amyloliquefaciens. Other studies have reported 31.88% and 25.96% increases in C. annuum and C. Chinese Jacq. plants through the inoculation of Bacillus sp. CKD061 and B. subtilis CBMT2, respectively43, 44. Likewise, the total fresh weight of C. annuum cv. Geumsugangsan seedlings increased by 27% with the treatment of B. amyloliquefaciens27. The morphological parameters are associated with vegetative development and improvement in crop yield45. Increased root length can enhance plant nutrition through larger absorption of water and nutrients46. Castillo et al. 47 mentioned that the increase in the fresh weight of poblano chili plants is associated with improved fruit quality. Otherwise, the inoculation of plants increased the concentration of photosynthetic pigments in chiltepin leaves. Hahm et al. 48 and Yasin et al. 26 showed increases of 28.19% and 29.46% in chlorophyll a in chili plants treated with B. iodinum KNUC7183 and the halotolerant isolate B. fortis SSB21. According to Mathivanan et al. 49, chlorophyll content is one of the leading indicators of photosynthetic and metabolic activity in plants. Furthermore, the accumulation of photosynthetic pigments in C. annuum var. glabrisculum is of particular importance during stages such as flowering and fruiting due to the high demand for photoassimilates for fruit formation50. Applying PGPR to the soil is an alternative to replace fertilizers and pesticides in horticultural crops51. These results suggest that the Bacillus spp. isolates could be used as a biofertilizer in chiltepin chili production.
Rhizobacteria exert a positive effect on plant growth by improving the availability of nutrients in the rhizosphere, producing molecules with chelating activity, and improving soil properties. The ability to solubilize phosphorus from its non-assimilable state (PO4 − 3) into its available forms (HPO4 − 2 and H2PO4−) has been reported in several soil microorganisms, including species of the genus Bacillus52. In this study, treatments showed an overall mean solubilization index of 2.84, similar results were reported by Mohamed et al. 53, who presented a solubilization index (SI) of 3.0 in B. subtilis. Other authors have studied the effect of B. megaterium to improve phosphorus plant nutrition in wheat plants exposed to low water availability10. In this report, a general mean of 2.81 was obtained in the zinc SI. These results are consistent with those reported in the literature; Naseer et al. 54 obtained an SI of 2.78. In another study, Bacillus sp. NCCP-49 presented a solubilization index of 2.8 when using the Bunt & Rovira medium supplemented with the same zinc source. Phosphorus and zinc solubilization are possible through medium acidification by releasing organic acids such as gluconic, oxalic, citric, lactic, tartaric, and aspartic acid. Also, it is attributed to the production of chelating agents and to the action of phosphatase enzymes that catalyze the hydrolysis of phosphate bonds55, 56. Otherwise, the NH4+ produced by rhizobacteria contributes to the nitrogen supply for plant nutrition and directly affects root development57. Wang et al. 58 observed higher biomass in chili plants inoculated with Bacillus isolates that produced ammonium and were isolated from saline soils. In this study, all Bacillus spp. isolates tested positive for the Nessler assay with 0.41 to 5.32 µmol of ammonium/mL. Similarly, Bhattacharyya et al. 57 presented NH4 + concentrations in the 2.5 to 7.54 µmol/mL range in Bacillus spp. Additionally, Abdelwahed et al. 59 conducted a microplate assay to reduce the Nessler reagent's environmental impact and toxicological risk. They observed a maximum concentration of ammonium of 371 and 370 µmol for B. inaquorsum and B. mojavensis, respectively. Another reported mechanism of action is the production of siderophores. These molecules of bacterial origin promote plant growth by improving iron availability to plants. Moreover, it is one of the main biological control mechanisms because PGPRs compete with phytopathogens for this essential element60. Bacillibactin is the predominant siderophore in B. subtilis, with a high capacity to chelate iron in extracellular media61. Consistent with the results of this study, Kazerooni et al. 27 found that all Bacillus spp. isolates isolated from soil produced siderophores. Additionally, EPS or bacterial-origin polymeric substances, are released as a response mechanism to physiological stress and represent a means of protection for both the host plant and the bacterium62. EPS produced by Bacillus spp. promote cell adhesion, soil aggregation, increase water permeability, enrich fertility, and enhance access to nutrient resources63. EPS production is also valuable for root colonization, as producing fibrous material favors adhesion to the root surface, especially under water scarcity conditions10. In conditions of water deficit, EPS produced by B. subtilis helps mitigate stress due to its hygroscopic and hydrophilic properties, which promote hydration and reduce desiccation64. In this study, all bacteria produced EPS with up to 2.53 g/L values. According to the categories described by Nwosu et al. 65, this result corresponds to a high capacity for EPS production, exceeding what was reported by Andy et al. 66, who observed high yields in isolates of B. cereus and B. haynesii with 1.8 and 1.5 g/L, respectively.
The rhizobacteria isolates displayed high tolerance up to an osmotic pressure of -1.75 MPa. Previous studies have reported varying degrees of drought tolerance in Bacillus spp. under similar conditions67. For instance, Ashry et al. 68 reported that 25% of isolates isolated from arid soils showed resistance to drought under different water potentials ranging from − 0.15 to -1.2 MPa. In another study, B. megaterium and B. licheniformis were able to grow at the lowest water potential tested (-0.73 MPa)10. This adaptation to water deficit is attributed to the genus ability of Bacillus to form endospores68. Other authors associate the resistance of Bacillus spp. and the amelioration of drought stress in plants with mechanisms such as EPS production, phytohormones, enzymes, and osmolytes10, 67.
According to the literature, bacterial treatments improve the survival of stressed seedlings. For instance, Kumar et al.69 demonstrated that treatment with B. pseudomycoides, B. massilioanrexius, and B. thuringiensis increased the survival rate of wheat plants subjected to water deficit with 10% polyethylene glycol (PEG) by 30.55%. In another study, B. amyloliquefaciens increased the survival rate of tomato plants under drought conditions by 55.29%. This effect was associated with bacterial biofilm production70. Additionally, Zhou et al.71 found that the ABA content was higher in stressed Arabidopsis thaliana plants inoculated with B. megaterium BOFC15, triggering the signaling of multiple adaptive responses. Besides the effect on survival, the Bacillus spp. isolates improved the symptomatology of chiltepin seedlings by reducing chlorosis in cotyledon leaves, stem deformation, necrotic areas in the radicle, and loss of turgidity. Other authors have reported mitigating water deficit in C. annuum plants using Bacillus spp. isolates27, 72. According to Arun et al.73, applying 25% PEG 6000 significantly reduces height and root length of rice seedlings. However, inoculation with B. megaterium PB50 increased the height by 26.37% and the root length by 46.91% in stressed plants. The authors also attributed the response to reduced transpiration rate and stomatal closure. Similarly, the effect of water stress on wheat plants with PEG (12%) was evaluated, and the isolates B. subtilis 104 and 26D increased the height by 7.89% in drought-sensitive varieties and 22.58% in drought-resistant varieties. Regarding root length, increases of 11.84% and 18.96% were reported in tolerant and sensitive varieties, respectively74. Otherwise, the reduction in the slenderness index indicated an improvement in the health condition of the seedlings. According to Borjas et al. 75, lower values correspond to plants with a greater capacity for adaptation to unfavorable environmental conditions.
Proline is considered osmoprotectants or osmolytes, small, neutral-charged molecules with low toxicity and a high capacity for water retention that accumulates in the cytoplasm76. These molecules can protect macromolecules and stabilize the membrane through hydrophilic interactions between proteins and lipids that compose it77. The water deficit causes a significant accumulation of proline; Kazerooni et al.27 showed a 64.1% increase in proline accumulation in C. annuum cv. Geumsugangsan plants treated with PEG (10%). However, in this study, the treatments showed less proline content in leaves compared to the negative control. This differs from what has been reported in the literature, as several studies show higher proline accumulation in inoculated plants as a resistance mechanism associated with maintaining cellular functionality27, 78. Considering the observed wilting symptoms, we conclude that there is mitigation of drought stress in chiltepin seedlings treated with Bacillus spp. through action mechanisms not reviewed in this study, such as antioxidant activity, hormonal, gene expression activation, and/or synthesis of protective proteins69, 10. For instance, Lozo et al. 79 demonstrated the mitigation of oxidative damage in C. annuum L. in response to drought with treatments using B. safensis SS-2.7 and B. thuringiensis SS29.2. These treatments reduced the accumulation of H2O2 by increasing the enzymatic concentration of ascorbate peroxidase, peroxidase, superoxide reductase, and glutathione reductase. Otherwise, RWC in leaves allows for quantifying the dehydration level of plants80, 81. In chiltepin, water retention caused a significant reduction of this parameter, which indicates a slowing of metabolic activity and growth inhibition due to the imbalance between the transpiration rate and water supply caused by the water deficit80, 78. According to categories described by Bandurska81, unstressed plants have an RWC in leaves above 90%, while plants under mild drought stress show values between 60 and 70%. An RWC between 40 and 60% indicates moderate water stress, and values below 40% correspond to severe stress. Thus, the treatment with Bacillus spp. reduced the stress level in chiltepin seedlings from severe to moderate. These results are consistent with the literature; Gupta et al. 6 reported a 15.47% increase in the RWC of C. annuum L. plants treated with B. amyloliquefaciens when subjected to drought stress (80% CC). Similarly, an increase of up to 38% in the RWC of tomato plants inoculated with B. subtilis Rhizo SF 48 was reported78. These results can be attributed to the conservation of leaf hydration through the modulation of stomatal behavior, decreased transpiration, reduced stomatal conductance, and photosynthetic81.
In conclusion, the Bacillus spp. from the soil originating from the central-south producing region of the state of Chihuahua favored germination and growth promotion of chiltepin under in vitro and greenhouse conditions. Furthermore, Bacillus spp. isolates mitigate the effects of water stress and increased resistance of chiltepin seedlings. B. cereus strain Bc25-7 stood out by increasing most of the vegetative parameters and tolerance to water stress in chiltepin. Remarkably, the application of the Bc25-7 isolate is a valuable alternative to contribute to the domestication of chiltepin and, with it, to in situ establishment through growth promotion and increased tolerance to water stress, using mechanisms such as ammonia production, siderophores, exopolysaccharides, zinc solubilization, and phosphorus.