Integrated Use of Plant Growth-Promoting Rhizobacteria and Chemical Fertilizers Improves the Growth and Yield of the Tomato Plant

Abstract

Microbial biofertilizers offer a sustainable alternative to reduce inorganic fertilizer inputs in intensive vegetable production. While rhizobia are traditionally associated with legumes, their co-inoculation with native rhizobacteria for non-leguminous crops like tomatoes remains under-explored. This study aimed to isolate native rhizobacteria compatible with Bradyrhizobium diazoefficiens NE1-65 and evaluate their combined effect on the tomato plant (var. max F1) under reduced inorganic fertilizer rates. From the initial eighteen isolates screened on nitrogen-free media, and solubilization assays of phosphorus and potassium, three isolates (RM-8, RM-17, RM-18) were found compatible with B. diazoefficiens NE1-65. Isolate RM-17 (tentatively identified as Aureimonas sp. based on 16S rRNA gene sequence) was selected for its high K-solubilizing capacity (KSI = 8.60). Then, a 90-day growth trial compared various fertilizer application rates (0, 25, 50, 75, and 100%) with and without the bacterial consortia. The 75% fertilizer rate plus the consortia significantly outperformed the 100% fertilizer rate alone. Specifically, it increased plant height (11.57%), fruit diameter (9.23%), fruit number (53.90%), and fruit weight (16.15%). These findings demonstrate that the RM-17 and B. diazoefficiens NE1-65 consortia can partially substitute inorganic fertilizers while significantly enhancing tomato growth and yield, highlighting its potential application for sustainable tomato production systems.

1. Introduction

Integrated fertilizer applications that combine chemical fertilizers and biofertilizers or bacterial consortia are one of the key strategies to reduce soil fertility decline brought about by injudicious and over-application of chemical fertilizers. Over the years, many have reported about microbial inoculation as a viable strategy for sustainable crop production, with many studies on legume inoculation. Thus, exploring the utilization of microbial inoculation for vegetables like the tomato may be a promising approach to reduce the dependence on chemical fertilizers.

In a review article, it was stated that several attempts to improve biological nitrogen fixation in non-leguminous plants such as maize were carried out and positive results were obtained [1]. Meanwhile, a study compared the inoculation of legume-associated Rhizobium with a non-legume-associated Azotobacter on the tomato plant, revealing that while there is a lesser yield with Rhizobium than Azotobacter inoculation, the antioxidant activity, lycopene, and vitamin C content was higher with Rhizobium inoculation [2]. Several plant growth-promoting rhizobacteria (PGPR) were evaluated in another study, where it was revealed that through various mechanisms, PGPR including Psuedomonas sp., Bacillus sp., Rhizobium sp., Azospirillum, Azotobacter, Enterobacter, Streptomyces sp., Proteobacteria, and Actinobacteria, contributed positively to the growth and yield of the tomato plant [3]. Meanwhile, co-inoculation of microbial inoculants comprising bacteria and fungi was tested for 3 years on field corn, revealing an increase in overall corn growth and yield and a reduction in the build-up of NPK in the soil [4]. Another report compared the utilization of a multi-species microbial inoculant composed of Agrobacterium, Azotobacter, Azospirillum, Bacillus, Pseudomonas, Streptomyces, Trichoderma, and Rhizophagus irregularis with chemical and mineral-based fertilizers, observing an increase in the overall growth of wheat with microbial inoculants [5]. This research demonstrates the beneficial effects of combined species compared with mineral-based and chemical fertilizers. On the other hand, a study used ten bacterial isolates from Enterobacter and Serratia and applied them in a dose-dependent manner on tomato seedlings. Better growth was observed as a result of phytohormones that were released by the isolates [6]. A similar result was reported when tomato plants were inoculated with four strains of Streptomyces, Priestia, Rossellomorea, and Psuedomonas and growth was compared against mineral fertilizers. The results indicated an overall growth increase in the inoculated plants [7]. Aside from co-inoculation of different rhizobia, combining beneficial microorganisms as inoculants with inorganic fertilizers proved to be a viable strategy to increase the yield of sugarcane [8] and is also reported to increase the yield of tomatoes [9]. Thus, this strategy could be used to reduce farmers’ dependence on inorganic fertilizers. These reports highlight the beneficial effects of using multiple species and/or strains as inoculants for various crops including tomato plants, which are sometimes greater or comparable with chemical fertilizers.

While rhizobia are traditionally recognized for their symbiotic N-fixing relationship with legumes, recent studies highlight their potential for enhancing the growth and productivity of non-leguminous crops such as tomatoes [10]. These beneficial bacteria promote plant growth through diverse mechanisms, such as biological N fixation, P solubilization, siderophore production, phytohormone synthesis, and suppression of soil-borne pathogens. In tomato plants, rhizobial inoculation has been reported to enhance nutrient absorption and improve root development [11], increase fruit yield and protect the crop from certain diseases and stress [12], and improve the plant’s tolerance to salt stress [13]. Although symbionts of legumes, bradyrhizobia share PGPR properties with many rhizobia, such as production of phytohormones and siderophores, and can colonize non-leguminous crops. Leguminous rhizobia, particularly Bradyrhizobium japonicum and Rhizobium, were reportedly used as plant growth-promoting rhizobacteria (PGPR) for a non-leguminous crop. In particular, the B. japonicum strain Tal629 was inoculated on radish plants and produced a significant increase in dry matter yield by as much as 15%. This is the first report that utilized bradyrhizobia in a non-leguminous crop [14]. The use of B. japonicum for tomato plants was also reported recently as a biocontrol agent for tomatoes, where it regulated the pathogenicity of Meloidogyne incognita and enhanced its growth [15]. So far, only B. japonicum species have been tested on non-leguminous crops. Meanwhile, B. diazoefficiens (formerly B. japonicum) has never been reported as an inoculant for vegetables. B. diazoefficiens USDA110 is a model strain that is used globally as a reference strain with N-fixation ability. Specifically, it has the nosZ gene that denitrifies N₂O as N₂ and it can modulate host interactions [16].

In the Philippines, researchers released their first report on locally isolated B. diazoefficiens NE1-65, which was used as an inoculant for soybeans. This report found that B. diazoefficiens NE1-65 increased the amount of N fixed on the soybean plant compared to the control [17]. As a locally isolated bacterium with high N-fixing ability, this isolate may be further explored for its use as an inoculant for vegetables. In addition, locally developed PGPR are better than imported strains since they are already adapted to local conditions. Further, it has been stated that locally developed PGPR are the preferable strategy due to their regional and plant-specific efficiency that is more sustainable than that provided by imported ones [18]. Thus, one aim of this study is to utilize locally isolated B. diazoefficiens NE1-65 as a reference strain and combine it in a bacterial consortium with newly isolated and local rhizobacteria from the tomato rhizosphere to develop local biofertilizers that are already adapted to local agro-climatic conditions.

The tomato plant (Solanum lycopersicum L.) is one of the most important horticultural crops worldwide, valued for its high nutritional content, economic relevance, and wide range of uses in both fresh and processed forms. In the Philippines, recent government initiatives encourage combined or balanced fertilizer application strategies involving both inorganic and biofertilizers for any crop due to severe soil degradation that is mainly attributed to the overuse of chemical fertilizers [19]. As tomato production relies heavily on inorganic fertilizer, the addition of biofertilizer to reduce the dependence on inorganic fertilizers may lessen soil degradation. Thus, this research is relevant because it addresses this pressing issue in soil degradation and contributes to existing knowledge about the combined application of PGPR and chemical fertilizers, not only in the Philippines, but also in countries where tomatoes are cultivated.

Therefore, this report focuses on (i) evaluating the use of B. diazoefficiens NE1-65 (a legume-associated rhizobacteria) as an inoculant for the tomato plant, in combination with a newly isolated rhizobacteria from the tomato rhizosphere, and (ii) determining the effect of the bacterial consortium in combination with different rates of fertilizer (0, 25, 50, 75, 100%) on the growth and yield of tomato plants grown in a mesocosm experiment.

2. Materials and Methods

2.1. Site Characterization, Soil Sampling, and Analysis

The soil used for bacterial isolation was collected from a organically managed vegetable cropland planted with eggplant, bitter gourd, tomatoes, finger pepper, and leafy vegetables that has been under organic management for nearly 30 years (since 1997). Tomatoes are routinely cultivated here from October to February. Organic management involves the use of compost every cropping season, which is applied basally at transplanting or seed sowing (10 t/ha). Organic soil amendments (6–10 kg/ha) and organic plant supplements (10 Li/ha) are also applied 2–3 times every growing season. Ten soil subsamples were randomly collected from a depth of 0–20 cm, thoroughly homogenized, and quartered to obtain a 1 kg composite sample. From this composite sample, a 100 g fresh soil sample was used immediately for bacterial isolation, while the remaining soil was air-dried, pulverized, and submitted to the Regional Soils Laboratory in San Fernando, Pampanga, Philippines for the analysis of the following soil physicochemical properties: texture (hydrometer method, soil pH, and EC (potentiometric 1:1 soil water)), total N (Kjeldahl method), available phosphorus (Olsen method), exchangeable potassium (leaching–flame AES), calcium (leaching–flame AES), magnesium (leaching–flame AES), sodium (leaching–flame AES), organic matter (Walkley and Black (colorimetric) method), cation exchange capacity (cation displacement–Kjeldahl distillation), copper (DTPA extraction–flame AAS), iron (DTPA extraction–flame AAS), manganese (DTPA extraction–flame AAS), and zinc (DTPA extraction–flame AAS).

2.2. Isolation and Characterization of Bacterial Isolates

For the isolation, 1 g of soil sample was subjected to a serial dilution up to 10⁻⁹. Aliquots (1 mL) from dilutions of 10⁻⁶ to 10⁻⁹ were spread onto freshly prepared yeast extract mannitol agar (YMA) plates supplemented with Congo red (0.025%) for the isolation of potential plant growth-promoting bacteria. Plates were incubated in the dark at 28 °C and monitored at 12 h intervals for colony development. The reference strain Bradyrhizobium diazoefficiens NE1-65, locally isolated from soybean root nodules in the Philippines and previously described was obtained as a pure culture grown on YMA slants and re-streaked on YMA plates alongside the newly isolated strains.

Dominant colonies exhibiting entire margins, creamy white pigmentation, convex elevation, and slightly liquid to mucoid consistency were selected and repeatedly streaked on YMA plates (4–6 successive subcultures) until pure cultures were obtained, as confirmed by the absence of Congo red uptake. The purified isolates were subsequently cultured in yeast mannitol broth (YMB) and incubated in the dark with continuous shaking at 120 rpm for 3–5 days until sufficient turbidity was achieved [20].

2.3. Screening for Plant Growth-Promoting Traits and Compatibility Testing

All isolates were screened for plant growth-promoting traits by culturing on Jensen’s agar for nitrogen fixation, Pikovskaya (PKV) agar for phosphorus solubilization, and modified Aleksandrow agar for potassium [21]. Growth on Jensen’s media was considered indicative of nitrogen-fixing ability, while the formation of a halo zone on PKV and Aleksandrow agars indicates the capacity to solubilize phosphorus and potassium, respectively. The phosphorus solubilization index (PSI) was calculated using the following formula [22]: phosphate solubilization index (PSI) = A/B, where A represents the total diameter (colony + halo zone) and B represents the colony diameter. The potassium solubilization index (KSI) was calculated using the same formula but as described in another report. Only isolates exhibiting high PSI and KSI values, positive growth on Jensen’s medium, and compatibility with the reference strain B. diazoefficiens NE1-65 were selected for further evaluation. The reference strain B. diazoefficiens NE1-65 was chosen because it was reported to have high N-fixation ability and was isolated from the same geographical region, although from a different site and land use system [23], to ensure microbial compatibility for biofertilizer development.

2.4. Molecular Identification and Phylogenetic Analysis

Genomic DNA was extracted from each culture using BL buffer according to the protocol reported [24] and based on the method described and cited earlier. The extracted DNA was used as a template for polymerase chain reaction (PCR) amplification of the 16S rRNA gene using primers reported in 1991 for bacteria [25]. The amplified PCR products were then submitted to Macrogen, Seoul, Korea for sequencing of the 16S rRNA gene. After that, a phylogenetic tree was constructed using the blastn report from the National Center for Biotechnology Information (NCBI) using Molecular Evolutionary Genetics Analysis (MEGA) v.12.12.

2.5. Plant Material and Pot Preparation

Tomato seedlings (Solanum lycopersicum L.) var. Diamante max F1 were grown under screenhouse conditions at the College of Agriculture, Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines (5°43′57.58″ N, 120°55′51.52″ E). Healthy 25-day-old seedlings were transplanted into sterilized pots, with one seedling per pot. Each pot was filled with 7 kg of field soil collected from the Ramon Magsaysay Center for Agricultural Resources and Environment Studies (RM-CARES) located at Central Luzon State University.

2.6. Preparation of Bacterial Consortium

Isolated bacteria were purified, and thereafter, colony morphology and functional characteristics were tested. After screening, compatibility testing with B. diazoefficiens NE1-65, and sequence analysis, the representative bacteria (PGPR) were selected to be co-inoculants. RM stands for Ramon Magsaysay, the location where the soil samples were collected, as previously described. Each isolate was cultured separately in YMB and incubated until an adequate cell density was achieved, as determined by optical density with a spectrophotometer (Seikohsya, Tokyo, Japan) at 600 nm (OD₆₀₀). Subsequently, equal volumes of PGPR and the B. diazoefficiens strain NE1-65 were mixed in a 1:1 ratio. The resulting mixture was then diluted with sterile distilled water to a final volume of 100 mL to form the bacterial consortium, following previously described methods [17]. The final bacterial suspension was adjusted to a concentration of approximately 1 × 10⁸ CFU mL⁻¹ before inoculation.

2.7. Experimental and Treatment Designs

The experiment was conducted in a greenhouse following a completely randomized design (CRD) with six treatments, each replicated three times. The treatments were as follows: T1: (control)—no application; T2: 100% inorganic fertilizer (60-0-90 kg N-P₂O₅-K₂O per ha); T3: 75% inorganic fertilizer + bacterial consortia at 10 mL plant⁻¹; T4: 50% inorganic fertilizer + bacterial consortia at 10 mL plant⁻¹; T5: 25% inorganic fertilizer + bacterial consortia at 10 mL plant⁻¹; T6: 0% inorganic fertilizer + bacterial consortia at 10 mL plant⁻¹. The rates of inorganic fertilizers were based on soil test recommendations and were applied in three splits at 7, 15, and 30 days after transplanting (DAT). The rhizobial consortia were applied twice at 7 and 14 DAT. Crop maintenance, including irrigation and pest management, followed standard conventional practices and was uniformly applied across all treatments.

Harvesting commenced at 60 DAT when the fruit reached physiological maturity, as indicated by the color change from pink to light red, and continued until the fourth priming. Plant height, fruit diameter, fruit weight per plant, and number of fruits per plant were recorded during the final priming at 90 DAT.

2.8. Data Analysis

All data were subjected to an analysis of variance (ANOVA) using R software (version 4.3.2), and mean comparisons were performed using Tukey’s honestly significant difference (HSD) test at a 95% confidence level.

3. Results

3.1. Soil Characterization

Soil samples for bacterial isolation were collected from a vegetable cropland at the RM-CARES, where a diverse range of crops has been cultivated under organic management throughout the year. The site has not received any inorganic fertilizers or pesticides since 1997, with nutrient inputs primarily derived from compost, organic fertilizers, and green manures. The soil has a clay loam texture and belongs to the Maligaya soil series, with a flat topography (<3% slope), good drainage, and a neutral pH (7.25). The soil exhibited high phosphorus content, moderate organic matter (3.42%), and moderate cation exchange capacity (14.76 cmol kg⁻¹), indicating a moderate level of fertility. Additional soil physicochemical properties are presented in

Table 1. Physicochemical characteristics of the experimental soil and the isolation site of rhizobacteria isolates.

Parameter	Unit	Value
Texture	–	Clay loam
pH	–	7.25
Electrical conductivity	mS cm−1	0.76
Phosphorus	mg kg−1	141.91
Potassium	cmol kg−1	1.42
Organic matter	%	3.04
Total nitrogen	%	0.16
Calcium	cmol kg−1	17.23
Magnesium	cmol kg−1	6.67
Sodium	cmol kg−1	0.26
Cation exchange capacity	cmol kg−1	14.76
Copper	mg kg−1	14.62
Iron	mg kg−1	29.98
Manganese	mg kg−1	2.78
Zinc	mg kg−1	11.95

.

3.2. Isolation and Characterization of Bacterial Isolates

Bacterial isolates were selected based on their ability to grow on a nitrogen-free (N-free) medium. All eighteen isolates exhibited growth on yeast mannitol agar (YMA) plates and displayed colony morphology typical of rhizobia, characterized by creamy white pigmentation, slightly mucoid consistency, entire margins, convex elevation, and circular shape. In comparison, Bradyrhizobium diazoefficiens NE1-65 presented circular colonies with creamy white pigmentation, entire margins, convex elevation, and a highly mucoid consistency. Growth on Jensen’s medium was evaluated according to colony formation time, where growth observed within 72 h was recorded as high N fixation (++) and growth detected after 72 h was considered moderate N fixation (+). Since Jensen’s medium is a nitrogen-free selective medium, more rapid colony development reflects a greater capacity to utilize atmospheric nitrogen as the sole nitrogen source, thereby indicating a higher nitrogen-fixing potential under the tested conditions. The results demonstrated that only seven isolates exhibited nitrogen-fixing potential, as evidenced by fast growth (++) on Jensen’s medium. These isolates were RM-1, RM-6, RM-8, RM-9, RM-13, RM-17, and RM-18. However, evaluation of other plant growth-promoting activities revealed that in the phosphorus solubilization assay, RM-13 demonstrated the highest phosphate solubilization index (PSI) of 4.66, followed by RM-18, while RM-9 produced the lowest PSI. Regarding potassium solubilization, RM-4 showed the highest potassium solubilization index (KSI) at 10.00, followed closely by RM-6 (9.90), while RM-2 exhibited the lowest KSI. Notably, B. diazoefficiens NE1-65 did not produce a halo zone in either the phosphorus or potassium solubilization assays, indicating the absence of detectable phosphate- and potassium-solubilizing activity under the conditions tested. Following compatibility testing with B. diazoefficiens NE1-65, the majority of the isolates were found to be incompatible as their growth was inhibited in the presence of the reference strain. Only three isolates—RM-8, RM-17, and RM-18—demonstrated compatibility. These results are summarized in

Table 2. In vitro plant growth-promoting traits of bacterial isolates and compatibility test with B. diazoefficiens NE1-65.

Isolate	N Fixation	Solubilization Index		Compatibility with B. diazoefficiens
		P	K
RM-1	++	2.27	8.80	No
RM-2	+	2.92	5.40	No
RM-3	+	2.37	6.10	No
RM-4	+	2.16	10.00	No
RM-5	+	2.08	8.00	No
RM-6	++	2.04	9.90	No
RM-7	+	2.60	6.10	No
RM-8	++	2.84	8.20	Yes
RM-9	++	1.69	8.10	No
RM-10	+	2.16	8.70	No
RM-11	+	2.62	7.50	No
RM-12	+	2.34	6.40	No
RM-13	++	4.66	6.60	No
RM-14	+	2.32	7.80	No
RM-15	+	2.40	8.90	No
RM-16	+	2.18	6.60	No
RM-17	++	3.47	8.60	Yes
RM-18	++	4.41	7.90	Yes
B. diazoefficiens NE1-65	++	--	--

Note: ++ = growth within 72 h, + = growth after 72 h.

.

3.3. Phylogenetic Tree Analysis

The three bacterial isolates (RM-8, RM-17, RM-18) were then identified based on 16S rRNA gene sequence analysis employing the Basic Local Alignment Search Tool (BLAST v.2.17.0). The blastn report indicates that the three isolates are clustered together. Therefore, only one isolate was used for the preparation of the consortium. In this case, RM-17 was selected since it had the highest KSI (8.60) among the three compatible isolates.

Shown in Figure 1 is the phylogenetic tree of the three rhizobacteria (RM-8, RM-17, RM-18) that showed compatibility with the reference strain B. diazoefficiens NE1-65. The three isolates are grouped with Aureimonas sp. but still cluster separately due to low homology.

Since it is debatable that even a 99.0% sequence homology guarantees that an isolate belongs to a specific genus [26], the isolates were labelled as rhizobacteria until other genetic loci were sequenced. B. diazoefficiens NE1-65 (LC386875) was not subjected to 16S rRNA sequencing in this study since it was obtained as a pure culture and its DNA sequence was available at the National Center for Biotechnology Information (NCBI) website.

3.4. Influence of Bacterial Consortia Application on the Growth and Yield of Tomatoes

Based on the 90-day growth period, the application of the 75% recommended rate of inorganic fertilizer, combined with the rhizobial consortia (T3), resulted in the highest values for several growth and yield parameters, including plant height (62.10 cm), fruit diameter (39.56 cm), number of fruits per plant (32.60), and fruit weight (37.34 g), as shown in Figure 2. The values are provided in Appendix A,

Table A1. Effect of co-inoculation with bacteria strain RM-17 and B. diazoefficiens NE1-65, combined with different rates of inorganic fertilizer, on plant height at 45DAT (first priming), fruit diameter, number of fruits per plant, and fruit weight of the tomato plant (Solanum lycopersicum L.) var Diamante max F1.

Treatments	Plant Height (cm) 45DAT	Fruit Diameter (cm)	Number of Fruits/Plant	Fruit Weight (g/Fruit)
T1—Control (no application)	55.20	30.36 b	13.27 c	21.71 b
T2—100% inorganic fertilizer	55.80	35.91 a	15.03 c	31.31 a
T3—75% inorganic fertilizer + bacterial consortia	62.10	39.56 a	32.60 a	37.34 a
T4—50% inorganic fertilizer + bacterial consortia	60.37	36.56 a	19.47 b	34.94 a
T5—25% inorganic fertilizer + bacterial consortia	59.27	35.55 a	29.40 a	35.63 a
T6—Bacterial consortia	61.33	37.26 a	16.47 bc	34.29 a

Different letter suffix indicates significant differences in the post hoc test using Tukey’s HSD at 95% confidence level using R software v. 4.4.2.

. These values represented increases of 11.57%, 9.23%, 53.90%, and 16.15%, respectively, compared with plants receiving the full recommended rate of inorganic fertilizer alone (T2). The differences were statistically significant. It should be noted that all treatments inoculated with the bacterial consortia exhibited significantly improved growth and yield parameters compared with the uninoculated control (T1). Notably, tomato plants receiving bacterial inoculation with reduced or no inorganic fertilizer application demonstrated growth and yield comparable to, or greater than, those supplied with the full recommended rate of inorganic fertilizer. This finding indicates that the bacterial consortia can partially compensate for reduced inorganic nutrient inputs while maintaining tomato productivity.

Overall, the inoculation of bacterial consortia (Aureimonas sp (RM-17) and B. diazoefficiens NE1-5) significantly enhanced tomato growth and yield under reduced inorganic fertilizer rates. Particularly, the combination of bacterial consortia with 75% RRIF optimized crop performance. This treatment increased fruit weight by 11.57% compared to 100% RRIF and by as much as 12.52% compared to the control. Moreover, the fruit diameter under the 75% RRIF + bacterial consortia increased significantly by 9.23% over the 100% RRIF and 23.26% over the control. Notably, this synergy significantly increased the number of fruits per plant by 54%, indicating that microbial inoculation can effectively offset a 25% reduction in the use of chemical fertilizers while improving productivity.

These improvements on the yield and growth of tomato plants with decreased inorganic fertilizer application provides a positive impact for sustainable tomato production.

4. Discussion

This study provides the first report on the use of a microbial consortium composed of native rhizobacteria and B. diazoefficiens NE1-65 for tomato cultivation. At the time of writing, only one study in the Philippines documented the use of Rhizobium inoculation on the tomato plant [27], thus highlighting the need to fill this research gap. The rhizobial strains used in this study were locally isolated from agricultural soils in Nueva Ecija, Philippines, emphasizing their ecological adaptation and potential suitability for local cropping systems. The use of locally isolated and developed microbial inoculants is preferred to avoid failure of this strategy. The inefficiency of microbial inoculation is caused by factors such as incompatibility with the host plant, unfavorable agro-environment, and low concentration [28]. Also, failure is attributed to the poor survival of the rhizobial strains in the soil due to competition with the resident or native soil microbial community [29]. Thus, this present report demonstrates the potential of this locally isolated bacterial consortia to be a candidate biofertilizer for tomato production.

The newly isolated RM-17 strain used in this study as a co-inoculant with B. diazoefficiens NE1-65 is clustered together with the two RM-8 and RM-18 isolates and has the closest homology with Aureimonas sp. Yet, 16S rRNA gene sequence results, even at 99% similarity, is not an absolute threshold to specify the genus or the species [30]. Even at a 99.3% to 99.8% similarity, inter-species variation occurs [31]. Therefore, the three isolates that were sequenced in this study are referred to as rhizobacteria strains until other genetic loci, including different functional, housekeeping, and chromosomal genes, are identified.

The results of this study demonstrated that co-inoculation with the bacterial consortia, combined with 75% of the RRIF, significantly enhanced tomato plant growth and yield compared with the full RRIF and the control. This finding highlights the potential of rhizobial consortia to partially replace synthetic fertilizers without compromising crop productivity. Similar results were reported in the past. In particular, it was revealed that microbial inoculation combined with inorganic fertilizer improved the fruit yield and the quality of the cherry tomato, which was attributed to the likely (not measured) production of indole-3-acetic acid [32]. B. diazoefficiens (formerly B. japonicum) is extensively studied and most widely used as a microbial inoculant for the soybean plant. It also occupies two distinct niches in the soil: microsymbionts of legumes and free-living diazotrophic bacteria [33]. This suggests that B. diazoefficiens can be used not only for legumes, but also for non-leguminous crops since it is able to fix N in the soil. Although the direct molecular and physiological mechanisms underlying the observed yield enhancement were not investigated in this study, the plant growth-promoting traits (PGTs) of rhizobacteria and Bradyrhizobium species have been extensively documented, as stated above. In addition, rhizobacteria have been widely reported to enhance crop growth and yield, and improve soil health through mechanisms such as increasing nutrient uptake, nutrient solubilization, phytohormone production, and siderophore secretion [34]. Moreover, improved plant growth has been associated with physiological processes stimulated by rhizobacteria, including siderophore-mediated iron acquisition and ethylene regulation via ACC deaminase activity [35]. The synergistic interaction among Bradyrhizobium, Aureimonas, Bacillus, and Streptomyces for chili pepper has previously been shown to regulate rhizosphere microecology through functional complementarity [36]. Although these physiological and biochemical parameters were not directly measured in the present study, it is plausible that similar mechanisms may have contributed to the observed improvement in tomato plant growth and yield following bacterial consortia inoculation. Therefore, further studies investigating nutrient uptake dynamics and physiological responses are necessary to confirm these proposed mechanisms. Such synergistic interactions among compatible rhizobacterial strains have also been reported to enhance nutrient use efficiency and plant productivity under reduced fertilizer inputs. Previous studies have demonstrated that co-inoculation strategies involving multiple microbial taxa can significantly improve tomato growth, yield, and stress tolerance. For instance, yield enhancement has been reported following co-inoculation with Bacillus spp. and endophytic bacteria [37], Trichoderma harzianum combined with mycorrhizal fungi [38], and Azorhizobium caulinodans with Piriformospora indica [39]. Moreover, microbial consortia have been shown to enhance plant tolerance to abiotic stresses such as nutrient limitation and drought, and to improve soil health, further supporting their role in sustainable crop production [40].

Despite many studies on microbial consortia for the tomato plant, this is the first report that utilized B. diazoefficiens combined with rhizobacteria. Though reported to have a high N-fixation ability, the improvement in the growth and yield of the tomato plant cannot be attributed solely to the B. diazoefficiens NE1-65 as it does not possess the ability to solubilize P and K, unlike RM-17. The tomato plant has a high requirement for P and K as these two macronutrients are critical to the overall growth and development of any crop. Rhizobacteria that are able to solubilize P and K can influence the nutrient availability in the soil and can increase the ability of the plant to tolerate or cope with stresses, thus also affecting the growth, yield, and overall plant health [41,42,43,44]. Therefore, the present study contributes novel insights into the broader applicability of legume-associated rhizobia beyond their traditional host range. This finding supports the hypothesis that rhizobia possess inherent plant growth-promoting traits that are not limited to symbiotic interactions with legumes, and that they can also benefit non-leguminous crops such as tomato plants, potentially through phytohormone production and improved nutrient availability [45].

Overall, this study demonstrates that co-inoculation with locally isolated rhizobial consortia represents a viable and sustainable approach for tomato production. By enabling reduced reliance on synthetic fertilizers without yield loss, this strategy aligns with the goals of sustainable agriculture and environmentally friendly nutrient management. The use of compatible microbial consortia composed of functionally diverse strains offers promising prospects for future biofertilizer development and large-scale application in vegetable production systems.

5. Conclusions

This study reports, for the first time, the utilization of B. diazoefficiens NE1-65 (originally isolated from soybean root nodules) combined with a newly isolated rhizobacteria strain RM-17 (from the tomato rhizosphere), as a bacterial consortium. The bacterial consortium is a promising potential biofertilizer candidate for the tomato plant, enabling a 25% reduction in inorganic fertilizer application without compromising plant growth and yield. Although the findings of this study are encouraging, it should be noted that the experiment was conducted under controlled pot conditions, and therefore, responses under field conditions may differ. The survival and activity of the microorganisms under field conditions is one limitation that has to be considered; thus, additional field experiments should be conducted in the future. Future research based on this study’s findings will include the characterization of other plant growth-promoting traits (phytohormones and siderophore production), the identification of other genetic loci (functional genes), and field experiments on multi-location and multi-season trials.

Nevertheless, these results establish an important foundation for the development of potential biofertilizers that integrate legume-associated rhizobia with rhizospheric bacteria for application in non-leguminous crops.