Utilization of Gluconacetobacter diazotrophicus in Tomato Crop: Interaction with Nitrogen and Phosphorus Fertilization

Abstract

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium capable of colonizing different host plants. This work evaluated the impact of Colombian native isolate G. diazotrophicus GIBI029 on tomato yield and quality in response to nitrogen and phosphorus fertilization levels. The experiment was conducted under semi-controlled conditions using a split-plot design with four replicates and ten plants per treatment. Variables assessed included fruit count per plant, fruit weight, average fruit weight, production per plant, and yield. Application of GIBI029 without fertilization resulted in a higher number and weight of fruits per plant across harvests (7.1 fruits, 509.2 g) compared to both the unfertilized control (4.8 fruits, 271.7 g) and with complete nitrogen and phosphorus fertilization (5.2 fruits, 288.8 g). The behavior of these variables were similar for GIBI029 and complete fertilization (7.0 fruits and 510.7 g per harvest). The highest yields were obtained with GIBI029 without fertilization (106.1 t ha⁻¹) and with full nitrogen and phosphorus fertilization (106.4 t ha⁻¹). Under the evaluated conditions, native G. diazotrophicus GIBI029 isolate could effectively improve tomato growth and yield in contrast to the controls. Based on these findings, the reproducibility of this behavior should be confirmed, and the mechanisms involved in the plant–bacteria interaction should be determined.

1. Introduction

Nitrogen is an indispensable nutrient for plant growth. Despite the substantial amount of nitrogen present in the atmosphere and soil, its availability to plants is limited, necessitating the application of this nutrient to crops [1]. Over the past 50 years, agricultural production has witnessed a tenfold increase, with a corresponding surge in the utilization of nitrogen fertilizers [2]. Concurrently, agricultural practices account for approximately 80% of the nitrogen released into the environment, contributing to adverse effects such as eutrophication [3], in addition to escalating production costs. Furthermore, the industrial processes employed in the production of nitrogenous chemical fertilizers consume significant quantities of fossil fuels, which are non-renewable resources. This consumption results in the release of carbon dioxide, a potent greenhouse gas, thereby contributing to global warming [2].

Phosphorus exists within the soil in both organic and mineral compound forms. However, the quantity of phosphorus readily available for plant uptake is significantly lower than the total phosphorus content present in the soil matrix. Consequently, the application of phosphorus fertilizers becomes essential to fulfill the nutritional requirements of crops. Nevertheless, the efficiency of phosphorus recovery from chemically synthesized fertilizers is notably limited. Only a fraction, ranging between 10% and 20%, of the applied phosphorus is utilized by plants during the year of application, as the majority of phosphorus is rapidly immobilized or fixed within the soil [4].

Gluconacetobacter diazotrophicus is recognized as a plant growth-promoting bacterium due to its inherent capabilities for biological fixation of atmospheric nitrogen, solubilization of phosphorus that is fixed within soils, and the production of indolic compounds [5]. This bacterium was initially isolated and characterized by Cavalcante and Döbereiner [6] from sugarcane, and it has since become one of the primary endophytic microorganisms employed as a model organism to investigate plant–bacteria interactions in non-leguminous plant species [7]. In the context of cultivation of economically important crops, it has been suggested that G. diazotrophicus holds substantial potential, largely attributed to the unique combination of its physiological characteristics, which are effectively expressed under conditions that closely resemble those found within the fluids of sugarcane, namely acidic pH and elevated sucrose concentrations [8]. While several studies have documented the presence of G. diazotrophicus and related growth-promoting effects in various crops, including sugar beet, carrot [9,10], radish, papaya, coffee [11], pineapple [12], sweet potato, taro, and cassava [13], as well as its general occurrence in many tropical and subtropical plants [14,15], further research is needed to elucidate its impact on commercially significant crops such as tomatoes.

Tomato (Solanum lycopersicum) is a dicotyledonous plant belonging to the Solanaceae family and the Solanum genus. This vegetable is the most extensively cultivated species and has nine related wild species. In 2023, global tomato production was estimated at 192,317,973 metric tons of fruit, harvested from approximately 5,412,458 hectares, resulting in an average global yield of approximately 35.53 metric tons per hectare [16]. Tomato cultivation is characterized by relatively high production costs. In middle-income countries like Colombia, tomato production represents a particularly risky agricultural activity, characterized by several key aspects: generally small and dispersed cultivation areas, intensive capital and labor requirements, substantial post-harvest losses, and significant phytosanitary challenges, among other factors. In particular, tomato cultivation in Colombia exhibits relatively low yields and limited technological adoption in production practices [17,18], such as the underutilization of environmentally friendly fertilizers, namely biofertilizers. Furthermore, a considerable portion of production expenses is allocated to fertility inputs, including costly chemical fertilizers, which can lead to severe environmental disturbances if applied excessively [19].

To reduce the production costs associated with tomato cultivation and to alleviate its negative environmental impacts, research is increasingly focused on exploring novel alternatives, such as microbial inoculants derived from plant growth-promoting bacteria [20] or plant growth-promoting rhizobacteria (PGPR) [21]. These bacteria possess the capacity to adapt, colonize, and persist within the rhizosphere, thereby promoting plant growth and development through their metabolic activities. Furthermore, they can significantly influence the bioavailability of essential nutrients, as exemplified by their ability to facilitate phosphorus solubilization and biological nitrogen fixation [22].

Studies have been conducted to assess the growth-promoting potential of G. diazotrophicus in tomato, such as that by Cocking et al. [23], who demonstrated that root meristem cells of tomato were intracellularly colonized by G. diazotrophicus strain 5541 UAP under gnotobiotic conditions. On the other hand, Luna et al. [24] studied the colonization and yield promotion of G. diazotrophicus PAL5 (also known as the ATCC 49037 strain) in tomato seedlings under gnotobiotic conditions, where it was evidenced that the bacterium colonizes lateral root emergence sites, root hairs, and stomata. In the same study, under greenhouse conditions, they found that plants inoculated with the bacterium significantly increased both the number and weight of fruit production compared to uninoculated controls. Botta et al. [25] evaluated G. diazotrophicus PAL5 under gnotobiotic conditions as well as in tomato seedlings, both individually and in a mixture, with Azospirillum brasilense, Herbaspirillum seropedicae, and Burkholderia ambifaria. During the in vitro experiment, G. diazotrophicus was the best isolate that colonized the entire tomato plant. A high number of bacteria was recovered in both roots (10⁸ CFU/g dry weight) and in the stem and leaves (10⁵ CFU/g dry weight). However, in seedlings during the in vivo experiment, G. diazotrophicus did not show good results in stimulating plant growth when applied individually. In response, Pallucchini et al. [26] investigated the effect of biological nitrogen fixation on growth promotion in a hydroponic culture of tomato plants by inoculating two isolates of G. diazotrophicus: a mutant with impaired nitrogen fixation (Gd nifD-) and a wild-type strain (Gd WT) under nitrogen-rich and limiting conditions. It was evidenced that the functional nifD gene is crucial for the optimal promotion of plant growth by G. diazotrophicus.

The application of high levels of fertilizers can significantly impact the interaction between bacteria and their host plants. This aspect has been evaluated in the case of G. diazotrophicus under different nitrogen and phosphorus fertilization regimes. In particular, nitrogen seems to act as a selective factor, as suggested by Caballero-Mellado et al. [27] and Kirchhof et al. [28]. Muthukumarasamy et al. [29] showed that nitrogen fertilization limited the isolation of G. diazotrophicus, with higher bacterial counts (from 10⁴ to 10⁷) resulting in plants without additional nitrogen fertilization. Likewise, Bueno dos Reis Junior et al. [30] noted that, in certain sugarcane genotypes, higher nitrogen doses reduced the population of G. diazotrophicus, indicating a genotype-dependent response. Moreover, several studies [31,32] have reported differential responses among sugarcane varieties and diazotrophic bacterial isolates regarding yield, nitrogen content, and nitrogenase activity under different nitrogen levels. Recent research has expanded these findings to include other crops. For instance, Tufail et al. [33] highlighted the role of G. diazotrophicus in mitigating combined drought and nitrogen stress in maize by enhancing biomass, chlorophyll content, and nitrogen uptake, while Van Long et al. [34] demonstrated that combining G. diazotrophicus inoculation with reduced nitrogen fertilizer maintained rice yield and improved soil quality in a triple rice cropping system. These studies indicate the potential of G. diazotrophicus to reduce nitrogen fertilizer inputs while sustaining crop productivity and soil health under diverse environmental conditions. Therefore, the study of the interaction between this bacterium, in conjunction with nitrogen fertilizer, and crops like tomato represents a relevant and ongoing research topic.

On the other hand, the interaction between G. diazotrophicus and phosphorus fertilization is a critical factor influencing plant–microbe dynamics and nutrient acquisition. Several studies have highlighted that phosphorus availability can significantly affect the colonization and activity of plant growth-promoting bacteria, including G. diazotrophicus. For instance, Delaporte-Quintana et al. [35] reported that phosphorus deficiency in strawberry plants was alleviated by inoculation with G. diazotrophicus, which increased phosphorus uptake and biomass by solubilizing inorganic phosphate. Similarly, Delaporte-Quintana et al. [36] showed that the PAL5 strain effectively solubilized different phosphate sources, supporting its functional role as a phosphate-solubilizing bacterium. Additionally, Idogawa et al. [37] demonstrated that high phosphate concentrations stimulated the production of levan, an exopolysaccharide that enhances stress resistance and colonization. These findings suggest that combining G. diazotrophicus inoculation with the addition of appropriate levels of phosphorus fertilizers may optimize nutrient cycling and contribute to sustainable crop production. Therefore, research on the role of this bacterium in relation to phosphorus fertilization, particularly in crops such as tomato, represents significant agronomic value.

Our research group has recovered a Colombian wild strain of G. diazotrophicus GIBI029, which has demonstrated notable plant growth-promoting properties, including nitrogenase activity, phosphate solubilization, and the synthesis of indolic compounds [38]. Furthermore, its potential in stimulating the growth of tomato seedlings (S. lycopersicum genotypes Santa Clara and Torrano) has been identified. Consequently, this study aimed to evaluate the interactive effects of inoculation with the G. diazotrophicus GIBI029 native isolate and varying doses of nitrogen- and phosphorus-based chemical fertilizers on tomato crop quality and yield. We hypothesized that the application of G. diazotrophicus GIBI029 native isolate will have a differential effect on tomato crop quality and yield compared to varying doses of nitrogen and phosphorus chemical fertilizers; it is expected that the bacterium can enhance yield even under reduced fertilizer input. This information will contribute to further establishing the behavior of this bacterium in tomato cultivation.

2. Materials and Methods

2.1. Location

The study was conducted at the Tesorito farm, Universidad de Caldas, situated in Manizales city (latitude 5°01′49″ N, longitude 75°26′13″ W), Colombia, in 2022 between February and September. The location experiences an average temperature of 17 °C, at an elevation of 2340 m above sea level (m a.s.l.), with an annual average rainfall of 1800 mm and a relative humidity of 78% [39]. The evaluation site was selected because it is one of the experimental farms of the Universidad de Caldas, which allowed for ensuring local control of the evaluated treatments. Additionally, the agroecological and climatic conditions of the geographical zone where the farm is located, above 2000 m a.s.l., have become ideal for the development of tomato cultivation under semi-controlled conditions, with neighboring associations of tomato producers identified as potential beneficiaries of extrapolatable and promising results.

2.2. Plant Material

A commercial Chonto-type hybrid tomato (Roble 956 F1 Hybrid Tomato, ImpulSemillas, Tocancipá, Colombia) was used, which is characterized by its good vigor and size, with strong stems and medium internodes. It is a material with indeterminate growth and a fruit weight between 140 and 160 g. It adapts to production areas with cold and medium climates (1800 to 2200 m a.s.l.), is tolerant to low temperatures, and resistant to pathogens such as nematodes, mosaic virus, and Verticillium or Fusarium 1, 2, and 3 [40]. The seeds used are certified and endorsed by a competent Colombian authority (Colombian Agricultural Institute) from the commercial company ImpulSemillas, under the commercial name “tomate Híbrido Roble” [41].

Planting was conducted in 128-cell nursery trays. Seedling production began with the disinfection of trays using agricultural iodine Agrodyne^® (West, La Estrella, Colombia) at a concentration of 5 mL per liter of water, in which the trays were submerged and then allowed to dry in the sun. Subsequently, the cells were filled with grade 4 peat-type substrate, avoiding compaction, and drilling holes were made with double the size of the seed for planting. Tomato seeds were then sown, one per cell, and the trays were placed in a germinator under semi-controlled conditions for roughly 30 days. Seedlings were kept in the germinator until they reached an average of 4 true leaves, indicating they were ready for transplanting. During the process, irrigation, fertilization, and pest control tasks were performed.

2.3. Microbial Cultures

Indigenous G. diazotrophicus isolation obtained in previous work [38], identified in the collection of microorganisms of the Universidad Católica de Manizales as GIBI029 (Manizales, Colombia), and the ATCC 49037 reference strain of G. diazotrophicus (ATCC, Manassas, VA, USA) were used. The bacteria were recovered from cryopreserved vials at −80 °C by reactivation at 37 °C for 5 min and subsequent sowing by depletion in Potato Dextrose Agar (PDA) (Oxoid^®, Waltham, MA, USA). The plates were incubated for 5 days at 30 °C until isolated colonies were obtained. From these cultures, the inoculations were prepared to be applied in the field.

2.4. Preparation of Greenhouses

The production system used was semi-controlled, which presented an indirect influence of external climatic variables. For this, two experimental semi-greenhouses were built. Total control was maintained only for the climatic variable of precipitation, managing the amount of water required by the plants through a drip irrigation system with emitters spaced at 20 cm. The temperature inside the greenhouses showed increases of between 2 °C and 4 °C in relation to the outside temperature. Black-black plastic mulch installed on the furrows was used to ensure weed control throughout the crop production cycle, thus avoiding a critical weed period that could alter the results. Additionally, the plastic mulch allows for moisture retention in the root zone because the evaporation process prevents the loss of water and nutrients. Constant humidity also provides an adequate microclimate for the inoculated bacteria, allowing for a more reliable measurement of the effect of soil application.

The soil in both greenhouses was prepared for tomato cultivation by adjusting the pH based on initial soil analysis (Table A1, Appendix A). Agricultural lime (240 g per linear meter) was manually incorporated using a hoe. Additionally, 360 g of organic matter was also incorporated into the soil. The land was leveled with a rake, and the irrigation hoses were further installed (see Figure 1).

2.5. Experimental Design

The experiment employed a split-plot design arranged in four randomized, completely replicated blocks. The main plot (main factor) corresponded to the application of G. diazotrophicus reference strain (ATCC 49037), the Colombian native G. diazotrophicus isolate (GIBI029), and the control for a total of 3 main plots (the control main plot was included in both greenhouses, as shown in Figure 2; for the statistical analysis, an equal and appropriate number of effective plants corresponding to this control main plot were taken from both greenhouses). Thus, the subplot (subfactor) comprised four levels of nitrogen and phosphorus fertilization. For this, each main plot was further divided into four smaller plots or blocks (n = 4 replicates), to which the four fertilization treatments were randomly assigned (see Table 1).

Each treatment combination (main plot × subplot) was applied to 10 individual plants. Each main plot, with its four replicates (blocks), contained a total of 160 plants (4 nitrogen and phosphorus fertilization levels × 10 plants × 4 blocks), i.e., a total of 480 plants in the 3 main plots. Consequently, each block consisted of 40 plants, representing a given bacterial treatment and all combinations of nitrogen and phosphorus fertilization. Each treatment was applied directly to 10 plants per combination per block.

The plants were established at a distance of 1.2 m between rows, spaced at 0.4 m between plants. In the corresponding main plot, 75 mL of a bacterial suspension of strain ATCC 49037 or isolate GIBI029 was applied as a drench to the base of each plant in the experimental units. The bacterial suspension was prepared from a bacterial culture with a concentration of 18 × 10⁷ CFU mL⁻¹, at a dose of 5 mL L⁻¹, and was inoculated 20 days after transplanting, as determined in previous works [42]. Four fertilization treatments were applied, arranged as illustrated in Figure 2 and Table 1.

2.6. Transplant and Crop Management

At 30 days post-germination, plants were transplanted from the nursery to the two experimental greenhouses. Seedlings were established at a spacing of 40 cm intra-row and 120 cm inter-row, resulting in a planting density of 20,833 plants ha⁻¹. Nutrient supplementation was tailored to the crop based on a comprehensive soil analysis of the experimental site (see Table A1 in Appendix A). All essential elements were supplied according to the species’ requirements, with the exception of nitrogen and phosphorus. These macronutrients were administered at either 100% or 0% of the recommended levels, interacting with the evaluated microorganisms as defined by the fertilization treatments outlined in the experimental design (Table 1). Additional micronutrients required by the crop were applied according to the soil analysis: MgSO₄ (IFFCO Kisan Suvidha Ltd., New Delhi, India) at a rate of 520 kg ha⁻¹, KCl (Lvfeng Fertilizer Co., Ltd., Zibo, China) at a rate of 937 kg ha⁻¹, and a commercial micronutrient mixture containing boron, copper, manganese, zinc, and iron (Yara International ASA, Oslo, Norway) at a rate of 205 kg ha⁻¹. A split application of fertilizers was carried out, starting at transplanting and continuing for 150 days, as presented in Table 1. All fertilizations were applied in a ring around each plant at the corresponding times and at the doses described in Table 1. To facilitate fertilizer dilution, continuous irrigation was ensured through drip irrigation lines with emitters spaced at 15 cm, one line per row, with a discharge rate of 25 mL min⁻¹. The amount of water applied per plant was 500 mL × plant⁻¹ × day⁻¹ from transplanting up to 30 days, 1000 mL × plant⁻¹ × day⁻¹ from 30 to 60 days, and 1500 mL × plant⁻¹ × day⁻¹ from 60 to 180 days (end of the cultivation period).

Crop architecture was managed by maintaining two stems per plant across all experimental units and standard pruning practices were implemented [43,44]. Upon reaching a total of 12 floral clusters on the main axis, apical bud pruning was performed to terminate vegetative growth and prevent further cluster development.

2.7. Evaluation of Yield and Quality

For each experimental unit of every replicate, the following variables were quantified: number of fruits per plant, weight of fruits per plant (in g/plant), mean fruit weight (in g), and total production per plant (in g plant⁻¹). Crop yield (in ton ha⁻¹) was subsequently estimated based on the determined production per plant and the established planting density. Fruit weight per harvest was recorded over a 10-week period, encompassing 10 distinct harvests throughout the crop cycle, with one harvest conducted per week. Fruits harvested weekly were those attaining a ripening index corresponding to either green-yellow (scale 2) or greenish-yellow (scale 3), as determined by visual fruit coloration according to the United States’ standards for grades of fresh tomatoes [45]. The summation of all partial harvests within each treatment provided the total production per plant (g plant⁻¹). The time leading up to the first harvest, specifically 90 days post-transplantation, was recorded, noting that the initial harvest and the first week of harvest coincided.

2.8. Statistical Analysis

Statistical analysis of the data was conducted using analysis of variance (ANOVA), employing the General Linear Model (GLM) procedure with SAS software version 9.4 (SAS Institute, Cary, NC, USA) to assess the effects of each experimental factor and their interactions. This analysis was performed to determine statistically significant differences among the various treatments. Where significant differences were detected, post hoc mean comparisons were carried out using Duncan’s multiple-range test at a significance level of p < 0.05.

3. Results

3.1. Number of Fruits per Plant

The impact of bacterial type (native isolation or reference strain) on tomato fruit number revealed statistically significant differences (p < 0.05) between the standard strain and the native G. diazotrophicus isolate in relation to varying nitrogen and phosphorus fertilization regimes throughout the crop cycle. This outcome is depicted in Figure 3, where the plants applied with the native isolate exhibited higher number of fruits per plant.

The GIBI029 isolate demonstrated superior performance across all interactions, exhibiting peak production during the second and fourth harvests (see Figure 4). Specifically, it yielded 11.2 fruits per plant in the second harvest under 100% nitrogen and 100% phosphorus fertilization, and it yielded 12.85 fruits per plant in the fourth harvest under nitrogen-free fertilization with 100% phosphorus. For the case when the native GIBI029 isolate was applied, statistical analysis indicated no significant differences (p < 0.05) in fruit number per harvest between varying nitrogen and phosphorus doses. Significantly, across all treatments and interactions, the highest mean fruit per plant value throughout the ten harvest cycles was observed in the N0P0 control interaction (7.1 fruits per plant) and in the N100P100 complete nitrogen and phosphorus fertilization treatment (7.0 fruits per plant), under the influence of the GIBI029 isolate. This suggests that the bacterium can function effectively utilizing inherent soil nutrient reserves, obviating the need for chemical fertilization when soil nutrient availability aligns with the requirements of the cultivated species.

The ATCC 49037 strain exhibited the lowest fruit number values across the experiments compared to the native isolate’s performance. The values recorded for this strain were consistently lower than those of the absolute control (nitrogen and phosphorus combinations without bacterial inoculation, see Figure 4). The interaction between nitrogen and phosphorus fertilization levels, along with the application of the ATCC 49037 strain, yielded peak fruit numbers when 100% of both phosphorus and nitrogen fertilizers were administered, exceeding the performance of the absolute control (N0P0). This observation underscores the dependence of this strain on external nitrogen and phosphorus sources to enhance fruit number production (Figure 4). For this variable, the efficacy of the GIBI029 isolate was evident in enhancing production outcomes within systems employing technical fertilization, thereby capitalizing on the utilization of native isolates.

3.2. Weight of Fruits per Plant

Concerning fruit production per harvest over time (Figure 5), statistically significant differences (p < 0.05) were observed across the majority of harvest periods for the two bacterial treatments and the controls. Notably, the tenth harvest exhibited no statistically significant differences between the two bacterial treatments. The peak harvest production was achieved 118 days post-sowing (fourth harvest). This harvest yielded values of 997 g plant⁻¹ and 356 g plant⁻¹ when utilizing the GIBI029 isolate and the ATCC 49037 strain, respectively. It was also found that the highest values were reported in the harvests 2, 4, and 5, respectively, and the lowest values were observed in the last three harvests 8, 9, and 10, respectively, for all the evaluated combinations, as depicted in Figure 5.

During harvests 4, 5, and 6, GIBI029 reported values of 997 g plant⁻¹, 846 g plant⁻¹, and 331 g plant⁻¹, respectively, while the control exhibited values of 555 g plant⁻¹, 354 g plant⁻¹, and 238 g plant⁻¹, respectively. Reference ATCC 49037 strain also showed statistically significant differences (p < 0.05) with values of 356 g plant⁻¹, 245 g plant⁻¹, and 231 g plant⁻¹ in harvests 4, 5, and 6, respectively (Table 2).

A similar trend to that observed for the number of fruits per plant was found for fruit weight over time (Figure 5). G. diazotrophicus GIBI029 isolate (in its different interactions with nitrogen and phosphorus fertilization levels) showed the highest values compared to the control and the reference ATCC 49037 strain. Nitrogen and phosphorus doses (0% and 100%) did not show statistically significant differences (p < 0.05) within each larger plot.

Throughout all harvest periods, the GIBI029 isolate consistently demonstrated a greater effect than the ATCC 49037 strain (Table 2). This phenomenon can be attributed to the fact that GIBI029 was isolated under agroclimatic conditions closely resembling those of the experimental site. This similarity likely facilitated the adaptation of the bacterium to the experimental conditions, thereby maximizing its potential for production enhancement.

The differential impact of four nitrogenous and phosphorous fertilization regimes on the per plant weight of fruits across the ten harvests in the tomato cultivation was observed at the seventh harvest, revealing statistically significant differences (p < 0.05) for the 0% nitrogen/100% phosphorus and 100% nitrogen/100% phosphorus treatments. The critical role of nitrogen application to reach the highest production was demonstrated by the superior production observed in treatments receiving 100% nitrogen. Specifically, at the seventh harvest, per plant weight of 451 g and 463 g were recorded for the N100P100 and N100P0 treatments, respectively, compared to 354 g for the unfertilized control (N0P0) and 286 g for the N0P100 treatment (Table 2). Evaluation of the interaction between the GIBI029 isolate, the ATCC 49037 strain, and the varied nitrogen and phosphorus fertilization levels yielded results consistent with those observed for fruit numbers across the progression of harvests. Notably, the GIBI029 isolate consistently exhibited the highest fruit weight across all nitrogen and phosphorus combinations throughout the crop’s lifecycle. In particular, the GIBI029 isolate demonstrated peak performance at the second, fourth, and fifth harvests, followed by the control and the ATCC 49037 strain treatments, with statistically significant differences observed (p < 0.05).

3.3. Mean Fruit Weight

Regarding the influence of bacterial type on the temporal dynamics of mean fruit weight, statistical significance was established (p < 0.05) across a majority of harvests, with the sole exception being the fourth harvest, where no significant divergence was observed between the two bacterial types applied. Throughout the sequential harvests, the GIBI029 isolate consistently exhibited a superior effect on mean fruit weight compared to the standard ATCC 49037 strain (Figure 6a).

The mean fruit weight, measured between 90 days after harvest and 167 days after transplanting, exhibited statistically significant differences (p < 0.05) for the GIBI029 isolate when compared to both the control group and the reference strain ATCC. While the initial harvests for GIBI029 yielded mean fruit weights exceeding 70 g and 75 g, respectively, a gradual decrease in weight was observed over time. Notably, the overall mean fruit weight for the GIBI029 treatment remained consistently higher than that of the control and the reference strain throughout the experimental period. It is important to clarify that the mean fruit weight reported here represents the mean of all harvested fruits, irrespective of fruit quality.

Analogously, during the second week (harvest), corresponding to 101 days post-transplantation (dpt), an increase in the mean fruit weight was observed. This augmentation was succeeded by a period of stabilization spanning the fourth and fifth harvests (123 dpt and 134 dpt), after which a progressive decline persisted until the termination of the cultivation cycle. This pattern evidenced the inherent tendency of the crop to exhibit a reduction in mean fruit weight over the duration of the harvest period (Figure 6a). Conversely, the influence of the four distinct nitrogen and phosphorus fertilization treatments on the mean fruit weight of the tomato crop, assessed throughout the eight harvests, did not show statistically significant variations (p < 0.05), except for the seventh and eighth harvests, specifically at 156 dpt and 167 dpt (Figure 6b).

3.4. Total Number of Fruits per Plant and Total Production per Plant

In general, the total number of fruits per plant across the 10 harvests (see Table 3) showed statistically significant differences (p < 0.05) for the bacterial application factor. The highest values were observed with the GIBI029 isolate, exceeding 65 fruits per plant by the end of the 10 harvests. In contrast, no significant differences (p < 0.05) were found among the smaller plots within each main plot, indicating that the bacterial treatment had a greater effect than the nitrogen and phosphorus fertilization levels. Specifically, the GIBI029 isolate outperformed both the control and the reference strain ATCC 49037. Furthermore, nitrogen and phosphorus fertilization at 100% levels, based on soil analysis, did not differentially affect fruit number in the smaller plots.

The cumulative mean value reached by the end of the last harvest (also called total production per plant) was 4862.4 ± 1268.8 g per plant for the GIBI029 isolate, followed by 2635.6 ± 937.5 g per plant for the controls (without bacterial application), and finally 2051.1 ± 854.6 g per plant for the ATCC 49037 strain. These results indicate an 84.5% increase of the total production per plant reached by the GIBI029 isolate related to the control mean value. In contrast, a 22.2% decrease of the total production per plant related to the controls was verified for the reference strain (Table 3).

Consistent with the preceding analysis of yield components (number of fruits per plant and fruit weight per plant), the best results of the total production per plant were found with the GIBI029 isolate that always exhibited values above 4500 g per plant, followed by the production achieved in the control treatments (without bacteria) and by the ATCC 49037 strain, with statistical differences (p < 0.05). Quantitatively, the GIBI029 isolate achieved production levels of 5107 g per plant under full nitrogen and phosphorus fertilization and 5092 g per plant in the absence of chemical fertilization, contrasting sharply with the ATCC 49037 strain under unfertilized conditions (N0P0), which yielded 1792 g plant⁻¹ (Figure 7a). The bacterium’s capacity to utilize soil phosphorus reserves, as reported in the soil analysis (see Appendix A), and its ability as a nitrogen fixer to ensure yields similar to those achieved with complete fertilization (100% nitrogen supplementation) according to the crop’s nutritional demand are evident.

In cases where one of these two elements was absent, such as the 0% phosphorus or 0% nitrogen treatments, production showed a slight downward trend, falling below 5000 g per plant in the presence of the GIBI029 bacterium. However, no statistically significant differences were observed at a 95% confidence level among the different interactions within this bacterium. In contrast, the values achieved for the control group and the reference bacterium ATCC fluctuated at around 3000 g per plant, showing statistically significant differences at a 95% confidence level compared to the GIBI029 bacterium across the different groups of the control and the ATCC strain in their respective interactions.

In the control treatment, the inherent response of the soil’s native nutrient reserves was evident. However, these reserves alone did not ensure increased production, highlighting the necessity of interventions to enhance nutrient availability. Within the control treatment group, nitrogen and phosphorus sources showed no statistically significant differences (p < 0.05), with values ranging from 2248.5 g plant⁻¹ (0% nitrogen, 100% phosphorus) to 2888.3 g plant⁻¹ (100% nitrogen, 100% phosphorus). In contrast, the ATCC reference strain, across its various nitrogen and phosphorus interactions, exhibited values below 2273.9 g plant⁻¹ (100% nitrogen, 0% phosphorus), reaching a minimum of 1792.4 g plant⁻¹ in the absence of both nitrogen and phosphorus (Figure 7a).

3.5. Crop Yield

To estimate the hectare-based yield (t ha⁻¹), we extrapolated from the plant density (20,833 plants ha⁻¹) and the mean production per plant (g plant⁻¹). This calculation revealed substantial differences among treatments, highlighting the potential impact on the production system’s sustainability. Specifically, the GIBI029 isolate, under varying nitrogen and phosphorus levels, resulted in yields exceeding 94.7 t ha⁻¹ (N0P100) and surpassing 100 t ha⁻¹ in the N0P0 and N100P100 treatments. In contrast, the ATCC reference strain exhibited significantly lower yields, ranging from a minimum of 37.3 t ha⁻¹ (N0P0) to a maximum of 47.4 t ha⁻¹ (N100P0) (Figure 7b).

Correspondingly, in the evaluation of crop yield per treatment, statistically significant differences (p < 0.05) were observed between the reference strain and the native isolate. Specifically, the GIBI029 isolate exhibited a yield of 101.3 tons per hectare, while the ATCC 49037 strain yielded 42.7 tons per hectare. This disparity signifies an augmentation of approximately 58.6 t ha⁻¹, representing a 137% increase in crop yield attributable to the application of the GIBI029 isolate (refer to Figure 7b and Table 3).

Regarding crop yield as a function of the four distinct fertilization regimes (Table 3), analysis revealed that the maximum yield was attained with complete nitrogen and phosphorus fertilization, registering a mean value of 67.9 tons per hectare. Subsequently, treatments featuring 100% nitrogen fertilization without phosphorus fertilization yielded 64.3 t ha⁻¹, closely followed by the treatment lacking both nitrogen and phosphorus fertilization, which yielded 64.2 t ha⁻¹. Conversely, the minimum yield was associated with the N0P100 fertilization regime, producing 57.5 t ha⁻¹.

During the examination of the interactive effects of the evaluated factors, namely bacterial type and nitrogen–phosphorus fertilization regimes, it was evident that combined treatments yielded values exceeding those obtained with individual factors. Specifically, the GIBI029 isolate, in conjunction with both N100P100 and N0P0 fertilization regimes, achieved a crop yield of 106 t ha⁻¹. These results demonstrated statistically significant differences (p < 0.05) when compared to the control treatments. The control treatments, which lacked bacterial inoculation, included the following variations in inorganic fertilization: no nitrogen and phosphorus (N0P0), full nitrogen fertilization without phosphorus (N100P0), full phosphorus fertilization without nitrogen (N0P100), and full nitrogen and phosphorus application (N100P100). These controls exhibited yields ranging from 46.8 t ha⁻¹ (N0P100) to 60.2 t ha⁻¹ (N100P100) when compared to the ATCC 49037 strain, which produced yields between 37.3 t ha⁻¹ (N0P0) and 47.4 t ha⁻¹ (N100P0). This observation underscores the synergistic potential of the GIBI029 isolate in combination with specific nitrogen and phosphorus fertilization regimes to substantially enhance crop yield.

These findings indicate the necessity of developing robust biological tools optimized for target environments to ensure beneficial interactions. The synergistic effects of such tools are crucial for achieving sustainable yields that surpass conventional agricultural practices, as demonstrated by the superior performance of the native GIBI029 isolate compared to a market reference strain. While reference strains may exhibit significant efficacy in their native habitats, their effectiveness can be diminished when transferred to different environments. Consequently, their inherent growth-promoting potential and cumulative impact on yield components may not be fully realized in productive systems, a limitation observed in this study with the market reference strain in contrast to the native GIBI029 isolation.

4. Discussion

4.1. Soil Analysis

The soil analysis reported a high phosphorus value of 299 ppm and low levels of calcium, magnesium, and potassium at 2.38 cmol kg⁻¹, 0.65 cmol kg⁻¹, and 0.19 cmol kg⁻¹, respectively. In contrast, the microelement values were within optimal average levels according to soil interpretation values reported by Molina [46]. The nutritional supplementation was tailored to the crop based on the soil analysis of the experimental site, the characteristics of which are detailed in Appendix A. According to IGAC and CORPOCALDAS [47], the soils in the Bellavista forest area, where the farm for the experiment is located, are composed of volcanic ash and are moderately weathered. They are soils with low-to-medium acidity, rich in organic matter, and have a medium-to-very high cation exchange capacity.

In general, the native GIBI029 isolate demonstrated the capacity to function effectively using the existing nitrogen and phosphorus levels indicated by the soil analysis (without supplemental nitrogen and phosphorus). Moreover, applying 100% nitrogen and 100% phosphorus did not change the bacterium’s effect, implying that nitrogen and phosphorus fertilization is not required when the soil contains sufficient reserves of these elements. In such cases, these elements can be mobilized by nitrogen-fixing and phosphorus-solubilizing bacteria, such as the native Gluconacetobacter isolate (GIBI029).

4.2. Fruit Number and Weight

Concerning fruit number, the GIBI029 isolate exhibited the most favorable performance among the bacterial treatments. In particular, across the ten harvests, the highest number of fruits per plant was consistently observed in treatments inoculated with the GIBI029 bacterium across the various combinations of nitrogen and phosphorus sources. This suggests a positive influence of GIBI029 on fruit number and, consequently, yield.

In the context of microbial inoculant evaluations, Hernández and Chailloux [48] and Luna et al. [24] documented 17.73 fruits per plant when inoculating tomato cultures with Glomus mosseae and Pseudomonas fluorescens under optimal treatment conditions. This observation contrasts with the results obtained in the present study, where the GIBI029 isolate yielded a mean value of 68.9 fruits per plant across the ten harvests. Luna et al. [24] demonstrated increased tomato fruit number and weight under gnotobiotic and greenhouse conditions using the G. diazotrophicus ATCC 49037 strain. However, unlike the results obtained in this work, their experiment did not explore responses across nutrient stress gradients. In the present study, the GIBI029 isolate exhibited a higher performance than the ATCC 49037 strain and showed significant yield benefits under both full and zero fertilization, underscoring the superior adaptability of the native isolate under local agroecological conditions.

Aguilar and Sánchez [49], in their study involving Santa Clara tomato plants inoculated with Azotobacter sp. at the time of transplantation and subjected to 50% of the recommended fertilization, reported 17 fruits per plant in their most effective treatment. This indicates the enhanced performance of G. diazotrophicus in tomato plants as well as the conditions selected for its application compared to the application of Azotobacter sp.

Reis et al. [50] have reported that fruit number in tomato plants can fluctuate in response to endogenous phytohormone production, particularly auxins such as 3-indoleacetic acid. In this regard, G. diazotrophicus is recognized for its capacity to fix atmospheric nitrogen and enhance the synthesis of phytohormones, including auxins [5,50].

4.3. Per Plant Production and Mean Fruit Weight

Concerning per plant production, a 156% increase was observed relative to the commercial control (N100P100 without bacteria) when utilizing the GIBI029 isolate (5107 g plant⁻¹). This finding aligns with the previously cited report by Aguilar and Sánchez [49], which documented a production of 2000 g plant⁻¹. In general, within each of the main factors or main plots (application of native isolate, reference strain, or control), a positive interaction was evident between the bacteria and the fertilization source.

The interaction that promoted fruit production the most was observed with the GIBI029 isolate. The highest values for this interaction occurred at the 100% nitrogen and phosphorus fertilization levels, as well as in the absence of added nitrogen and phosphorus. It is important to clarify that the zero level of nitrogen and phosphorus refers to the absence of added fertilizer sources, but these plots still contained the residual nutrient levels reported in the soil analysis (see Appendix A). The control treatments, representing conditions without bacterial inoculation and varying levels of inorganic fertilization, yielded intermediate plant weights exceeding 2500 g. This observation demonstrates the validity of the control treatments selected for the experimental setup disclosed in this work since this intermediate value is consistent with the generally reported fruit production range of 2000 to 3000 g per plant in conventional tomato production systems [17]. This suggests that even without the introduction of bacterial treatment, standard fertilization practices in our experimental setup were capable of achieving yields within the typical range for traditionally cultivated tomatoes.

Specifically, for the GIBI029 isolate, the positive impact of G. diazotrophicus was confirmed through an observed increase in mean fruit weight. This augmentation in mean fruit weight has been documented in other studies involving tomato [24] and papaya [51]. The results obtained revealed no statistically significant differences in mean fruit weight among treatments with varying fertilization levels (p < 0.05), which aligns with findings reported by Aguilar and Sánchez [49] in tomato crops subjected to moderate and complete fertilization regimes.

4.4. Crop Yield

The application of a suspension of G. diazotrophicus GIBI029 isolation to the tomato crop demonstrated a positive effect on both fruit production and yield. This beneficial influence is also observed in the inoculation of other crops, including sugarcane, carrot [9,10], sugar beet [10], radish, coffee [11], and pineapple [12], utilizing this microbial species, where production increases ranging from 30% to 80% above control treatments have been documented. Likewise, the introduction of various bacteria from Gluconacetobacter genus to diverse crops has shown the potential to enhance yield, as evidenced by yield increases in taro (38%) [13], papaya (38%) [14], and cassava (45%) [13].

In this study, treatments with 100% nitrogenous fertilization levels exhibited the highest mean fruit number per plant. This observation aligns with the findings of Aujla et al. [52] and Direkvandi et al. [53] in their respective studies on tomato plants, where increasing nitrogen levels from synthetic sources resulted in an elevated fruit count per plant. Furthermore, Jaramillo et al. [44] reported that nitrogen is a fundamental factor in flower and fruit production and in the regulation of maturation. Similarly, the highest production per harvest during the production cycle was achieved with treatments receiving 100% nitrogen fertilization. In this context, Rojas et al. [5] emphasized the importance of nitrogen in tomato crop yield, as elevated nitrogen concentrations promote flower and fruit development. However, regarding final production, the maximum yield for the tomato crop was obtained with a balanced fertilization regimen (100% nitrogen and 100% phosphorus). Jaramillo et al. [44] asserted that balanced fertilization is crucial for successful greenhouse tomato cultivation. Direkvandi et al. [53] also observed the highest yields in the treatment with the highest nitrogen fertilization rate (225 kg N ha⁻¹), reaching approximately 4796 g tomato plant⁻¹. Additionally, Hernández and Chailloux [48] reported a yield increase of 300 kg ha⁻¹ when implementing a balanced fertilization strategy (100 kg nitrogen per hectare, 25 kg phosphorus per hectare, and 50 kg potassium per hectare) compared to fertilization with only 100 kg nitrogen per hectare.

In comparison to the overall Colombian yield (96 tons per hectare) and the regional yield for Caldas region (70 t ha⁻¹) in tomato crops cultivated under semi-controlled conditions [54], the application of the GIBI029 isolate enables an increase exceeding 31 t ha⁻¹ above the regional yield, thus approaching the national value.

The yield performance of the tomato crop was enhanced by the application of the G. diazotrophicus GIBI029 isolate. The yield exhibited a 168.3% increase related to the control with balanced fertilization (N100P100 with no bacteria), corresponding to a mean value of 101.3 t ha⁻¹, which can be compared to the findings of Alfonso and Galán [55], who achieved 32.7 t ha⁻¹ in their optimal treatment utilizing co-inoculation of mycorrhizae and rhizobacteria. This treatment involved the application of a mixture of Azospirillum brasilense and Glomus clarum, supplemented with 30 kg nitrogen per hectare (in the seedbed) and 60 kg nitrogen per hectare (in the field). Hernández and Chailloux [48], in their study, attained 34.3 t ha⁻¹ in their most effective treatment by inoculating the tomato crop with Glomus mosseae. Consequently, the singular application of G. diazotrophicus demonstrates the potential to yield superior outcomes for the growth promotion of tomato crops.

4.5. Harvest-Stage Analysis

The graph depicting the total production achieved in grams per plant over time, across different treatments (Figure 7a), illustrates a positive cumulative trend throughout the 10 harvests. This trend was evident in both the number of fruits and the weight of fruits obtained with each harvest. Specifically, the values reported for the bacterium GIBI029 across its various interactions reached up to 5000 g per plant for the 100% nitrogen and 100% phosphorus treatments, as well as the 0% nitrogen and 0% phosphorus treatments.

It is worth highlighting that fructification reaches its maximum production period between 90 and 100 days after transplanting. Subsequently, it begins to decrease until the end of the crop cycle. The genotype used has a harvest initiation time between 75 and 90 days after transplanting. This is a normal phenomenon over time. The initial harvests have the highest average fruit weight values, which decline as the number of harvests progresses. From the results obtained, it was evidenced that the GIBI029 isolate consistently exhibited a higher performance across the sequential harvests on mean fruit weight compared to the standard ATCC 49037 strain (Figure 6a).

This study reports statistically significant improvements in yield components (fruit number, total fruit weight, average fruit weight, and yield) when using the GIBI029 isolate. In addition to the cumulative yield improvements observed, the results obtained suggest that the influence of the native GIBI029 isolate varies across the crop cycle. For instance, peak fruit production and weight were observed with the GIBI029 isolate in the earlier harvests (specifically, harvests 2, 4, and 5), with a gradual decline in fruit weight in later harvests. During this period, plants treated with the native GIBI029 isolate consistently outperformed both the uninoculated controls and the plants inoculated with the reference strain ATCC 49037. This temporal behavior indicates that the beneficial impact of GIBI029 is closely associated with the early and mid-reproductive phases of tomato development, likely mediated by peak biological nitrogen fixation, enhanced phosphate solubilization, and increased production of indolic compounds (e.g., auxin) during critical stages of fruit set and early fruit development. This can be explained by the fact that the native G. diazotrophicus GIBI029 isolate may enhance nutrient mobilization (particularly, of nitrogen and phosphorus) early in the reproductive phase, supporting the development of a high number of fruits and increased fruit weight. In fact, other diazotrophs such as Azospirillium sp. have the ability to enhance nutrient availability in the rhizosphere [56]. As the plant matures and the demand for nutrients increases, the isolate’s capacity to sustain the same level of nutrient supply may become limiting; this may contribute to the decline in fruit weight in later harvests. On the other hand, the plant’s sink strength (the capacity of fruit to attract resources) changes over time. Early in development, there are fewer fruits, and they act as strong sinks. Later, with a higher number of fruits, the sink strength of individual fruits may decrease, naturally leading to smaller fruit size [57]. Finally, the activity of GIBI029 itself might be influenced by the plant’s physiological stage, root exudate composition [58], or changing environmental conditions within the rhizosphere over time [59].

While our study demonstrates the positive impact of GIBI029 on overall yield, the complex interactions between the plant and GIBI029 likely involve stage-specific dynamics that justify further investigation. Future studies could incorporate detailed plant physiological measurements (e.g., nutrient uptake, photosynthesis rates, hormone levels) at different growth stages, rhizosphere dynamics (e.g., root exudate analysis, microbial community changes, nutrient availability), and gene expression analysis to obtain an understanding of the mechanisms underlying the temporal patterns observed in the data presented in this work.

4.6. Overall Comparison of Results on Yield Components

The significant improvements in yield components observed with the native G. diazotrophicus isolate GIBI029 align with previous reports of growth promotion by endophytic diazotrophs but also present notable contrasts. Compared to Cocking et al. [23], our results demonstrate tangible agronomic benefits under semi-controlled greenhouse conditions, advancing from the earlier focus on colonization mechanisms. Likewise, Luna et al. [24] found that inoculation with the ATCC 49037 strain increased fruit number and weight under greenhouse conditions; however, their study did not test nutrient-limited conditions. In contrast, our findings show that GIBI029 outperformed ATCC 49037 and even full nitrogen and phosphorus fertilization, suggesting that strain origin and soil adaptation are critical. This indicates that native strains like GIBI029 may unlock greater yield potential in low-input systems than previously reported with standard reference strains. Botta et al. [25] found that the ATCC 49037 strain was ineffective alone in promoting tomato seedling growth in vivo. This sharply contrasts with GIBI029’s robust performance as a standalone inoculant, suggesting that local environmental adaptation is key to microbial success in real-world applications. Our results underscore that effectiveness is strain- and context-specific. Further contrast arises with Hernández and Chailloux [48] and with Aguilar and Sánchez [49], who reported lower maximum tomato yields (32.7 t ha⁻¹ and 34.3 t ha⁻¹, respectively) with co-inoculation of mycorrhizae and rhizobacteria, compared to the 106.1 t ha⁻¹ achieved with GIBI029 isolate even without synthetic fertilization. This suggests GIBI029 may offer greater efficiency in nutrient mobilization, particularly under conditions with moderate soil fertility and high organic matter. This superior performance may also relate to GIBI029’s higher nitrogenase activity, phosphate solubilization index, and indole compound production compared to the ATCC 49037 strain. These differences indicate that microbial inoculants can be effective alternatives to chemical fertilizers if properly selected for environmental and crop compatibility. Notably, GIBI029’s ability to maintain yield in nutrient-limited soils highlights its potential for reducing costs and environmental impacts, especially for smallholders and organic farming.

4.7. Comparison of Native Isolate and Reference Strain

This study successfully highlighted the differential effects observed when utilizing a regional isolation compared to a foreign reference strain. G. diazotrophicus ATCC 49037 strain (the same PAL5 strain) was used in this experiment since it has become a key endophytic bacterial strain employed as a model organism to investigate plant–bacteria interactions in non-leguminous plant species [7]. Furthermore, this strain has been evaluated in tomato seedlings in studies such as Luna et al. [24], where its plant growth-promoting potential has been demonstrated. Moreover, in seedbed studies, the application of strain ATCC 49037 at a dose of 2.5 mL L⁻¹ with added phosphorus to Santa Clara tomato seeds at sowing has been identified as a promising strategy for enhancing seedling growth. These assessments, focused on the seedbed stage, evaluated the vegetative development of the tomato plants [42]. For this reason, it was selected for inclusion as the reference strain of the same species as the native isolate used in the experimental design of this study.

On the other hand, the Colombian wild isolate of G. diazotrophicus GIBI029 exhibited a nitrogenase activity of 0.010 nmol of reduced acetylene × mL⁻¹ × h⁻¹, production of indole compounds of 78.5 µg × mL⁻¹, and a solubilization index for tricalcium phosphate of 3.619, as demonstrated in earlier in vitro assays [38]. The physiological capabilities of the GIBI029 isolate, which were reinforced by this study, suggest a more efficient mobilization of soil nutrients and greater auxin production, potentially contributing to its superior performance compared to other strains. This contrasts with the lower nitrogenase activity and phosphate solubilization capacity reported for the reference ATCC 49037 strain, as also observed in the comparative assays performed.

In this study, the GIBI029 isolation demonstrated superior performance compared to both the uninoculated control and the reference ATCC 49037 strain. This phenomenon should be analyzed in light of the results obtained during comparative in vitro assays on plant growth-promoting properties conducted in previous work [38]. The native GIBI029 isolate was recovered from the stem tissue of sugarcane plants cultivated in soil characterized by a substantial content of organic matter (ranging between 10.0% and 20.4%), acidic to slightly acidic pH values (between 4.4 and 5.9), and considerable nitrogen levels (between 0.40 and 0.68%). These inherent soil properties of the GIBI029 origin site are quite similar to the soil conditions in the experimental greenhouses used in this study (0.53% nitrogen, pH of 4.8, and 14.1% organic matter, as presented in Table A1 of Appendix A). The performance of this isolation can be considered as promising, taking into account that the bacterium was successfully applied to a dicotyledonous vegetable crop (tomato) different from the grass species (sugarcane) from which it originally was recovered.

The reasons for the low performance of this reference strain remain unclear. Several factors could explain this reduced performance. The ATCC 49037 strain, which was originally isolated from Brazilian sugarcane cultivars [6], may not be optimally adapted to the specific and unique agroecological conditions prevalent in the West Central region of Colombia, where this study was conducted. This can potentially limit its colonization and plant growth-promoting properties. In this sense, the best results observed with GIBI029 correlate strongly with the comparatively lower performance exhibited by the reference ATCC 49037 strain during the aforementioned in vitro assays [38], which showed a nitrogenase activity of 0.005 nmol of reduced acetylene × mL⁻¹ × h⁻¹ (half of the activity displayed by GIBI029), a production of indole compounds of 64.3 µg × mL⁻¹, (about 82% of the production observed with GIBI029), and a solubilization index for tricalcium phosphate of 3.097 (approximately 86% of the solubilization capacity of GIBI029). Although standard procedures were used for strain revival, there exists the possibility of some degree of deterioration in the ATCC 49037 strain during storage or subculturing, which might have affected its viability or effectiveness. In this regard, it is worth highlighting that this strain has been preserved by cryopreservation at −80 °C through a monitoring and custody process by the collection in which it is stored. In addition, differences in specific plant–microbe interactions between the tomato cultivar used and the two types of G. diazotrophicus applied (native isolate or reference strain) could also contribute to the observed differences in performance. It is worth highlighting that this study did not specifically investigate these factors, and therefore, these explanations remain speculative. Further research is needed to elucidate the specific reasons for the differential performance of GIBI029 and ATCC 49037.

Consequently, these elevated values of plant growth-promoting indices explain why the native GIBI029 isolate exhibited the most pronounced effects on the yield component variables of the tomato crop assessed in the present work, irrespective of nitrogenous or phosphorous fertilization.

4.8. Advantages of Native Colombian GIBI029 Isolate

The presented findings on the native Colombian G. diazotrophicus GIBI029 isolate, which significantly improved tomato yield and fruit production align with and extend prior research on the plant growth-promoting effects of this bacterium in different crops. The results obtained corroborate the colonization capacity and positive growth effects of this species observed under gnotobiotic conditions in the studies by Cocking et al. [23] and Luna et al. [24], both of which demonstrated the ability of G. diazotrophicus to enter tomato root tissue and enhance fruit yield components. However, our study extends this understanding by confirming the performance of the native Colombian GIBI029 isolate under semi-controlled greenhouse conditions, showing consistent fruit and yield increases even in the absence of synthetic nitrogen and phosphorus fertilization, a context not explored in those earlier works.

In contrast to Botta et al. [25], who found that G. diazotrophicus performed poorly under in vivo conditions when applied alone to tomato seedlings, this study provides robust evidence that the native GIBI029 isolate performs strongly when applied independently in soil systems. This highlights the importance of local adaptation and suggests that strain origin plays a critical role in determining inoculant efficacy.

The findings disclosed in this work expand upon the work of Ríos Rocafull et al. [10], who demonstrated that the plant growth-promoting effects of G. diazotrophicus on crops such as carrot and sugar beet are highly dependent on the type of culture medium used for inoculum preparation. While they observed variable responses depending on the carbon and nitrogen sources in the medium, our study used a standardized inoculum preparation (PDA-based) and still achieved consistent and robust effects on tomato yield across all fertilization regimes. This suggests that the native GIBI029 isolate may have enhanced adaptability or a more efficient interaction mechanism with tomato plants under semi-controlled conditions. Additionally, while these authors reported moderate biomass gains in short-cycle crops, the results of this study indicate that GIBI029 isolate significantly boosts yield components such as fruit number and fruit weight, achieving values over 100 t ha⁻¹ even in the absence of nitrogen and phosphorus fertilization. This contrast highlights the importance of host specificity and environmental context, indicating that GIBI029 may be particularly suited to Solanaceous crops and the agroecological conditions of the West Central Colombian highlands.

On the other hand, Pallucchini et al. [26] demonstrated the importance of functional nitrogen fixation genes in G. diazotrophicus for growth promotion in hydroponic tomato, aligning with our observation that the native GIBI029 isolate, which has shown a high nitrogenase activity, significantly improved fruit yield even without nitrogen and phosphorus fertilization.

4.9. Limitations of This Study

Although the present study clearly demonstrated that inoculation with the native G. diazotrophicus GIBI029 isolate significantly improved tomato yield and fruit production, it is important to recognize certain experimental limitations. Specifically, the rhizosphere microbial community structure and the colonization patterns of the bacterial isolates within the plant roots were not directly assessed. Consequently, while the results suggest that the observed benefits can largely be attributed to the strain-specific advantages of GIBI029, the possibility that unmeasured biotic interactions or shifts in the native microbiome contributed to these outcomes cannot be entirely excluded.

However, several elements within this study reinforce the hypothesis that the beneficial effects observed are predominantly due to intrinsic characteristics of the GIBI029 isolate. As noted above, previous in vitro characterization revealed that GIBI029 exhibits a higher nitrogenase activity, a greater capacity for phosphate solubilization, and enhanced production of indolic compounds relative to the reference ATCC 49037 strain. Furthermore, GIBI029 was isolated from agroecological conditions highly similar to those of the experimental site, including soil pH, organic matter content, and nitrogen availability. This environmental congruence likely contributed to the higher adaptation and performance of the native isolate under the conditions evaluated. Moreover, the consistent superiority of GIBI029 across all fertilization regimes, including treatments without nitrogen and phosphorus supplementation, indicates a robust growth-promoting effect that appears independent of external chemical inputs. This suggests that the advantages conferred by GIBI029 are primarily linked to its physiological capabilities rather than potential indirect effects mediated by shifts in the soil microbial community.

The semi-controlled conditions within the greenhouses where the experiment was conducted may have influenced why some treatments, such as the reference strain, did not exhibit the expected responses. The region’s average temperatures of 17 °C could have affected the bacteria and, consequently, their behavior. Furthermore, this study has areas for improvement, including the need to integrate physiological, molecular, and ecological analyses to clarify how the GIBI029 isolate promotes growth and whether its performance remains stable across different environments and cultivation systems. These considerations will be taken into account in the design of future research.

Nevertheless, as recognized in this study, future research efforts should incorporate a more comprehensive evaluation of the plant–microbe interaction mechanisms as well. Specifically, integrated approaches involving rhizosphere microbiome profiling through metagenomics, visualization of colonization patterns using fluorescently labeled strains, and transcriptomic analyses of both plant and bacterial gene expression during the interaction are necessary. These investigations will be critical to elucidate the functional basis of the observed plant growth promotion and to definitively exclude the contribution of unmeasured biotic interactions.

4.10. Comparison with Findings from Previous Studies

The present study confirms that the G. diazotrophicus GIBI029 isolate, combined with nitrogen and phosphorus fertilization (or without additional fertilization of these two macronutrients), enhances tomato yield and quality of the fruits. This aligns with prior research under macro-tunnel systems [60], where tomato yields increased by 28% and profitability improved by 35% with inoculation under 50–75% fertilization regimes.

The microbial effectiveness observed here is supported by prior work on the design of the culture medium for the production of G. diazotrophicus cell suspension, where the influence of pH and sucrose-based media was modeled to improve viability and bacterial cultivation performance [61]. That study demonstrated biomass yields up to 5.3 g L⁻¹ and stable nitrogenase activity under bench-scale conditions, ensuring viability and consistency for field application.

Comparable findings were reported in carrot cultivation, where inoculation with G. diazotrophicus under 75% nitrogen and phosphorus fertilization led to yield increases of 29% and higher root quality compared to full fertilization without inoculation [9]. The economic evaluation of that system showed profitability improvements of up to 22% [62], consistent with our results for tomato under open-field conditions.

In tomato seedlings, earlier results revealed significant interactions between G. diazotrophicus, plant genotype, and phosphorus fertilization levels, with shoot biomass increases of 30–45% under low-phosphorus conditions in the cultivars [42]. These findings support the results obtained in the present work and highlight the importance of genotype-specific responses to microbial inoculants.

Furthermore, plant growth-promoting properties such as nitrogen fixation, phosphate solubilization, and production of indole compounds have been previously identified in isolates of G. diazotrophicus (including GIBI029) and G. sacchari, recovered from Colombian sugarcane cultivars [38]. Such physiological traits provide a mechanistic basis for the improved nutrient efficiency and yield observed in this and the previous studies.

These findings collectively strengthen the evidence that G. diazotrophicus can sustainably enhance tomato production while reducing chemical fertilizer use.

4.11. Future Research

The superior performance of the native G. diazotrophicus GIBI029 isolate in this study undoubtedly suggests its adaptation to the specific agroecological conditions of the West Central region of Colombia. To fully elucidate the underlying physiological and molecular mechanisms responsible for these beneficial effects, further research is required. Specifically, our ongoing work includes a genomic analysis aimed at assessing biological nitrogen fixation through the identification of nif genes within the GIBI029 genome. Additionally, transcriptomic studies will be essential to identify the genes activated during the establishment of the interaction between this bacterium and the tomato plant. Another promising avenue for future investigation involves classical biochemical assays utilizing marked bacterial mutants, enabling the visualization of the bacterium’s colonization patterns within the plant through fluorescence microscopy. The comprehensive insights gained from these future research efforts will be crucial for strengthening the scientific evidence required to definitively define and recommend the native GIBI029 isolate as a valuable microbial inoculant, not only for vegetables like tomatoes but also potentially for carrots and other economically important crops in similar agroecological zones.

Results obtained in this work indicate that the native GIBI029 isolate holds promise as a potential commercial inoculant. However, it is crucial to recognize that this work did not consider its formulation stability, field reproducibility, and regulatory feasibility. The current study was conducted under semi-controlled greenhouse conditions, and several key aspects need to be addressed before moving towards commercial application. Research is required to determine appropriate formulations (e.g., liquid, solid, encapsulation) that ensure the stability and viability of GIBI029 during storage and application. Multi-location field trials under diverse agroclimatic conditions are necessary to validate the reproducibility of our results and the consistency of the effects of GIBI029 isolate in real-world agricultural settings. A thorough cost–benefit analysis is essential to evaluate the economic viability of using GIBI029 as a bioinoculant compared to traditional fertilization practices. The regulatory requirements for registering and commercializing a microbial inoculant in different regions must be carefully considered.

Therefore, while this study provides a strong foundation for further research, claims regarding its immediate commercial application would be premature. Future work should focus on addressing the points mentioned above and determining the precise mechanisms by which GIBI029 promotes plant growth to optimize its application and ensure consistent results.

5. Conclusions

Under sandy loam soil conditions, the application of the native Colombian G. diazotrophicus isolate GIBI029 to tomato cultivars exhibited a positive effect on plant growth compared to unfertilized controls. Using GIBI029 without nitrogen and phosphorus fertilization led to higher yields than those obtained with full conventional fertilization. This study revealed that G. diazotrophicus GIBI029 positively influenced tomato development throughout the growing cycle, significantly affecting key yield components, including fruit count per plant, fruit weight per plant, mean fruit weight, and total production per plant. The resulting final yields were substantial, reaching 5092 g per plant and 106 t ha⁻¹.

To fully understand the mechanisms by which GIBI029 enhances tomato plant growth and to assess the consistency of its performance across diverse environments and cropping systems, future research should incorporate integrated physiological, molecular, and ecological analyses. Based on the findings of the present study, these future investigations will be crucial for advancing our understanding of G. diazotrophicus’s potential in promoting economically significant crops like tomato. Consequently, a thorough understanding of these aspects could enable the formulation of an effective microbial inoculant for tomato production in tropical agricultural systems.

Author Contributions

Conceptualization, N.C.-A.; methodology, S.P. and J.A.C.; software, N.C.-A.; validation, G.M.R. and N.C.-A.; formal analysis, G.M.R., N.C.-A., and Ó.J.S.; investigation, S.P. and J.A.C.; resources, G.M.R., N.C.-A., and Ó.J.S.; data curation, N.C.-A. and S.P.; writing—original draft preparation, S.P., J.A.C., and N.C.-A.; writing—review and editing, Ó.J.S.; visualization, S.P. and J.A.C.; supervision, N.C.-A. and G.M.R.; project administration, G.M.R.; funding acquisition, G.M.R. and Ó.J.S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.

Acknowledgments

The authors thank the technical support of the Tesorito Farm at the Universidad de Caldas and the Research Institute in Microbiology and Agro-industrial Biotechnology at the Universidad Católica de Manizales.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1 Preparation of the greenhouses for the experiment setup, illustrating (a) the initial soil preparation and (b) the installation of irrigation hoses at 20 cm spacing and black plastic mulch over the planting rows to control weeds and retain moisture, which created semi-controlled conditions for the tomato cultivation and bacterial inoculation study.

Figure 2 Schematic diagram of the split-plot experimental design conducted within two greenhouses, demonstrating the arrangement of bacterial treatments (main plots) and fertilization levels (subplots or smaller plots) to assess their interactive effects on tomato yield. The design facilitated the comparison of GIBI029 and ATCC 49037 against controls under varying nutrient conditions. Solid colored lines represent rows receiving different edaphic fertilization treatments. The dotted plum-colored line depicts the bamboo stalks used for structural support, dividing each greenhouse into two main plots. Codes corresponding to the percentages of nitrogen and phosphorus fertilization are deciphered in Table 1.

Figure 3 Main plots within the two experimental greenhouses used in this study. (a) The main plot treated with the G. diazotrophicus ATCC 49037 reference strain (left) and the corresponding control main plot (right). (b) The main plot treated with the native G. diazotrophicus GIBI029 isolate (left) and the corresponding control main plot (right). GIBI029-treated plots visibly show denser and more productive plants than the controls, suggesting enhanced growth performance from the native isolate. Images were captured at the time of the fourth harvest.

Figure 4 Effect of G. diazotrophicus and four types of fertilization on the mean fruit number per plant across sequential harvests of the greenhouse tomato crop. Tomato plants treated with the native isolate GIBI029 consistently produced a significantly higher number of fruits across all fertilization regimes, with peaks during the 2nd and 4th harvests. This indicates a strong early-to-mid-cycle effect of GIBI029 on fruit set, independent of chemical fertilization. The codes corresponding to the percentages of nitrogen and phosphorus fertilization are deciphered in Table 1. ATCC: G. diazotrophicus reference strain (ATCC 49037); control: conventional control treatment; GIBI029: native G. diazotrophicus isolation; Harvest 1–10: number of tomato fruits per plant in each harvest (the numerical value indicates the harvest number). Treatments with different letters are significantly different (p < 0.05) according to Duncan’s multiple-range test (n = 4 replicates or blocks).

Figure 5 Effect of G. diazotrophicus and four types of fertilization on the weight of fruits per plant across sequential harvests of the greenhouse tomato crop. GIBI029-treated plants showed higher fruit weights in early and mid-harvests (notably harvests 2, 4, and 5), outperforming both the control and ATCC 49037 treatments. The effect was consistent even in the absence of nitrogen and phosphorus fertilization, highlighting the isolate’s growth-promoting potential. The codes corresponding to the percentages of nitrogen and phosphorus fertilization are deciphered in Table 1. ATCC: G. diazotrophicus reference strain (ATCC 49037); control: conventional control treatment; GIBI029: native G. diazotrophicus isolation. Treatments with different letters are significantly different (p < 0.05) according to Duncan’s multiple-range test (n = 4 replicates or blocks).

Figure 6 (a) Mean fruit weight over time by bacterial treatment; GIBI029 application resulted in higher average fruit weights, especially in early harvests, with a gradual decline over time, mirroring normal crop senescence. (b) Effect of four types of fertilization on the mean fruit weight (g) over time in the greenhouse tomato crop. Differences were minor across treatments, except during later harvests (7th and 8th), suggesting bacterial influence outweighed fertilization effects on this variable. ATCC: G. diazotrophicus reference strain (ATCC 49037); control: conventional control treatment; GIBI029: native G. diazotrophicus isolation. The codes corresponding to the percentages of nitrogen and phosphorus fertilization are deciphered in Table 1. Different letters indicate significant differences between treatments (p < 0.05) according to Duncan’s multiple-range test, with the figure showing both the main plot effect (a) and the subplot effect (b), where n = 4 replicates or blocks.

Figure 7 (a) Effect of the type of G. diazotrophicus bacteria applied and four types of fertilization on the total production per plant in the greenhouse tomato crop. GIBI029 treatments achieved the highest cumulative fruit production, exceeding 5000 g per plant even without nitrogen and phosphorus fertilization, demonstrating its robust effect on overall productivity. (b) Effect of the type of G. diazotrophicus bacteria applied and four types of fertilization on the yield in the greenhouse tomato crop. Yields exceeded 100 t ha⁻¹ with GIBI029, matching or surpassing those achieved with full fertilization, indicating potential for reducing chemical inputs without compromising productivity. ATCC: G. diazotrophicus reference strain (ATCC 49037); control: conventional control treatment; GIBI029: native G. diazotrophicus isolation. The codes corresponding to the percentages of nitrogen and phosphorus fertilization are deciphered in Table 1. Error bars show standard deviation (n = 4 replicates or blocks). Significant differences between treatments (p < 0.05) are indicated by different letters, as determined by Duncan’s multiple-range test.

Table 1

Scheme of edaphic fertilization during the crop cycle according to the treatment, detailing the specific treatments, fertilization sources, and application rates.

Treatments	Degree of Fertilization	Code	Fertilization Source	Days After Transplanting[g per Plant]					Dosage[kg ha−1]
				0	30	60	90	150
1	0% N + 0% P	N0P0	-	-	-	-	-	-	0
2	0% N + 100% P	N0P100	TSP	8.0	4.0	4.2	-	-	338
3	100% N + 0% P	N100P0	Urea	8.0	4.0	4.0	-	-	335
4	100% N + 100% P	N100P100	TSP	8.0	4.0	4.2	-	-	338
			Urea	8.0	4.0	4.0	-	-	335
1–4			Micronutrients	2.0	2.0	2.0	2.0	2.0	205
1–4			KCl	6.0	6.0	12.0	12.0	9.0	937
1–4			MgSO4	5.0	5.0	5.0	5.0	5.0	520

TSP: triple superphosphate.

Table 2

Comparative Duncan’s multiple-range tests for production indexes over time during the evaluation of G. diazotrophicus addition to the tomato culture.

Variable	Factor		Harvest
			1	2	3	4	5	6	7	8	9	10
Number of fruits per plant	Type of bacteria	GIBI029	5.25 a	11.94 a	3.32 b	12.38a	11.38 a	5.72 a	8.35 a	4.78 a	3.49 a	2.18 b
		ATCC	4.31 b	4.98 b	3.97 a	5.95c	4.23 c	4.13 a	6.33 b	2.65 b	1.24 c	2.77 b
		Control	3.28 c	3.65 c	2.97 b	8.17b	6.19 b	4.73 b	7.33 ab	4.10 a	1.99 b	5.65 a
	Type of fertilization	N0P0	3.93 a	5.85 a	3.11 a	8.13a	7.27 ab	5.62 a	6.41 b	4.20 ab	2.57 a	4.37 a
		N0P100	4.11 a	6.19 a	3.35 a	8.29a	6.30 b	4.54 ab	5.79 b	3.22 b	2.05 a	3.32 a
		N100P0	3.93 a	5.95 a	3.23 a	8.75a	6.69 ab	5.03 ab	8.92 a	4.87 a	1.930 a	3.73 a
		N100P100	4.15 a	6.23 a	3.52 a	9.49a	7.73 a	5.62 a	8.22 a	3.35 b	2.17 a	4.84 a
Weight of fruits [g per plant]	Type of bacteria	GIBI029	418.0 a	940.0 a	260.0 a	996.8a	846.2 a	331.0 a	487.2 a	275.4 a	189.9 a	117.9 b
		ATCC	207.0 b	240.0 b	191.0 b	356.1c	244.9 c	230.9 b	321.8 b	114.0 c	43.6 c	101.7 b
		Control	207.0 b	240.0 b	191.0 b	554.8b	354.0 b	238.8 b	372.5 b	177.4 b	81.3 b	218.8 a
	Type of fertilization	N0P0	259.8 a	415.0 a	208.3 a	591.8a	492.1 a	238.6 a	353.6 b	209.8 ab	116.8 b	195.4 a
		N0P100	259.8 a	415.0 a	208.3 a	580.4a	396.9 a	246.9 a	286.4 b	146.2 c	87.1 a	131.1 a
		N100P0	259.8 a	415.0 a	208.3 a	602.2a	419.3 a	260.3 a	462.9 a	229.3 a	87.4 a	142.6 a
		N100P100	259.8 a	415.0 a	208.3 a	688.0a	490.8 a	293.6 a	451.0 a	158.8 bc	104.4 a	187.9 a

Remarks: Within each column, means followed by different letters indicate statistically significant differences (p < 0.05). Differences may be attributed to either fertilization treatments (subplot effect; n = 4 replicates or blocks) or bacterial treatments (main plot effect). The codes corresponding to the percentages of nitrogen and phosphorus fertilization are deciphered in Table 1. ATCC: G. diazotrophicus reference strain (ATCC 49037).

Table 3

Cumulative values of total number of fruits per plant, total production per plant, and overall yield at the end of the tenth harvest, according to the bacterial treatment and nitrogen and phosphorus fertilization levels.

Variable	Factor	Treatment	Total Value
Total number of fruits per plant	Type of bacteria	GIBI029	69.0 a ± 16.5
		ATCC	40.5 c ± 15.5
		Control	48.0 b ± 16.1
	Type of fertilization	N0P0	49.9 bc ± 19.0
		N0P100	47.1 c ± 18.0
		N100P0	53.1 ab ± 17.2
		N100P100	55.3 a ± 21.6
Total production[g per plant]	Type of bacteria	GIBI029	4862.4 a ± 1268.8
		ATCC	2051.1 c ± 854.6
		Control	2635.6 b ± 937.5
	Type of fertilization	N0P0	3081.3 a ± 1579.4
		N0P100	2758.2 b ± 1365.7
		N100P0	3087.2 a ± 1294.4
		N100P100	3258.1 a ± 1620.5
Yield[t ha−1]	Type of bacteria	GIBI029	101.3 a ± 26.4
		ATCC	42.7 c ± 17.8
		Control	54.9 b ± 19.5
	Type of fertilization	N0P0	64.2 a ± 32.9
		N0P100	57.5 b ± 28.4
		N100P0	64.3 a ± 26.9
		N100P100	67.9 a ± 33.7

Remarks: Mean values with different letters in the column are significantly different (p < 0.05). Standard deviation is presented following the total values. Fertilization treatment comparisons (subplot effect), and bacterial treatment comparisons (main plot effect) are both represented (n = 4 replicates or blocks). The codes corresponding to the percentages of nitrogen and phosphorus fertilization are deciphered in Table 1. ATCC: G. diazotrophicus reference strain (ATCC 49037).

Appendix A

Table A1

Analysis of the initial soil sample of the Tesorito farm at the Universidad de Caldas.

Parameter	Units	Value
N	%	0.53
pH		4.8
Na	cmol/kg	0.43
Ca	cmol/kg	2.38
Mg	cmol/kg	0.65
K	cmol/kg	0.19
P	ppm	299
Fe	ppm	669
Cu	ppm	7.97
Zn	ppm	28.65
Mn	ppm	38.35
B	ppm	1.9
S	ppm	25.54
OM	%	14.08
Texture	Sand—76%; Silt—13%; Clay—11%

OM: organic matter.

Table A2

Mean comparison tests for the effect of nitrogen and phosphorus fertilization and two types of G. diazotrophicus on the content of nitrogen and phosphorus in a tomato-cultivated soil 180 days after transplanting under controlled conditions.

Type of Treatment	Nitrogen (%)	Phosphorus (mg/kg)
N0P0	0.61 a	220.33 b
N0P100	0.59 a	223.67 b
N100P0	0.58 a	273.67 a
N100P100	0.60 ab	259.22 ab
ATCC 49037	0.60 b	227.33 b
GIBI029	0.64 a	284.75 a
No bacteria	0.56 c	220.58 b

Remarks: Values with different letters in the column are significantly different (p < 0.05).

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