Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue

Abstract

Beneficial soil microorganisms, particularly plant probiotic bacteria (PPB), play a pivotal role in promoting plant growth, development, and overall health through root colonization. PPB-based biofertilizers offer a sustainable and eco-friendly alternative to conventional agricultural inputs. This study evaluates the plant probiotic potential of three native bacterial strains Rhizobium sp. ACO-34A, Sinorhizobium mexicanum ITTG R7^T, and Sinorhizobium chiapasense ITTG S70^T to enhance the growth, quality, and sugar content of Agave tequilana. A comprehensive genomic and functional analysis was conducted for each strain to assess their plant probiotic traits. Additionally, a greenhouse inoculation assay was performed on six-month-old agave seedlings at the “piña” stage to evaluate the effects of these strains on plant growth and sugar content. Comparative genomic analysis revealed that these rhizobial strains harbor genes associated with key plant probiotic traits, reinforcing their role in enhancing plant development. The results demonstrated significant effects (p < 0.05) on growth and sugar content in inoculated plants. ACO-34A increased plant height by 35.4%, fresh weight by 41.5%, and inulin content by 57.3%, while ITTG-R7^T showed improvements of 26.4%, 35.2%, and 38.2%, respectively, compared to the control, and ITTG S70^T also exhibited enhancements, although to a lesser extent, with increases of 23.5% in plant height, 28.9% in fresh weight, and 31.2% in inulin content. These findings highlight the biofertilizer potential of these native rhizobial strains, particularly Rhizobium sp. ACO-34A, positioning them as promising candidates for the sustainable cultivation of A. tequilana.

1. Introduction

Agave tequilana, commonly known as blue agave, holds significant economic, social, and cultural importance in Mexico, primarily as the raw material for tequila production. With increasing national and international demand, tequila production has grown into a multibillion-dollar industry, generating substantial employment opportunities and making a significant contribution to the economy of Mexico, with exports rising at an annual average rate of approximately 7.5% [1].

Beyond tequila production, A. tequilana exhibits great potential in the food, medicinal, and agroindustrial sectors, largely due to its high content of inulin-type fructans, including short-chain fructooligosaccharides (scFOS), oligofructose, galactooligosaccharides, lactulose, and bioactive compounds such as phenolic acids and flavonoids with prebiotic properties that promote gut health [2,3]. These fructans are increasingly valued as low-calorie sweeteners, fat substitutes, and dietary fibers used in the formulation of functional foods [4], such as cereal-based products [5], dairy-based foods such as yogurt [6], and bakery products [7]. Additionally, they offer therapeutic benefits, such as blood glucose regulation, lipid profile improvement, and immune function modulation, highlighting the importance of A. tequilana in the nutraceutical and medicinal fields [8].

Despite its significance, A. tequilana cultivation faces challenges. Its long growth cycle (8–10 years) and reliance on substantial chemical fertilization present environmental risks, including heavy metal accumulation in soils, degradation of soil structure, and compromised long-term fertility [9]. Sustainable alternatives, such as biofertilization, are therefore essential to meet agave production demands while protecting soil health and preserving environmental integrity. Beneficial soil microorganisms, particularly plant probiotic bacteria (PPB), are emerging as eco-friendly options to reduce reliance on chemical fertilizers while enhancing crop productivity and quality in a sustainable manner [10].

Plant probiotic bacteria (PPB) are beneficial microbes that colonize plant roots, promoting growth and health through mechanisms such as phytohormone production, nitrogen fixation, phosphate solubilization, and secretion of antibacterial and antifungal metabolites [11]. By positively interacting with their host plants, PPB enhance crop yields and quality while reducing the need for synthetic fertilizers and pesticides [12]. Additionally, PPB contribute to biofortified foods by boosting bioactive compounds beneficial to human health, such as antioxidants and fructans in A. tequilana, positioning these bacteria as promising biofertilizers [13,14]. PPB differ from plant growth-promoting bacteria (PGPB) by their strict requirement for non-pathogenicity to humans and plants, a criterion critical for food safety. This restricts PPB to specific bacterial genera such as Rhizobium, Azotobacter, Azospirillum, and a few species of Bacillus, which exhibit both safety and efficacy in plant growth promotion [9]. PGPB, including plant growth-promoting rhizobacteria (PGPR), are a subgroup of PPB that primarily focus on growth enhancement and stress mitigation [15].

Among the plant probiotic bacteria (PPB), Rhizobium and Sinorhizobium are particularly versatile and effective in promoting plant growth. Traditionally known for their nitrogen-fixing symbiosis with legumes, Rhizobium species have also demonstrated significant growth-promoting effects in non-leguminous crops, enhancing nutrient uptake, disease resistance, and overall crop quality [16]. Several studies have reported the plant probiotic potential of Rhizobium strains in various crops. For instance, Rhizobium leguminosarum PETP01 and TVP08 in pepper, R. laguerreae PEPV40 in spinach, and R. leguminosarum PEPV16 in lettuce and carrot have shown improved plant growth, nutrient absorption, and fruit quality, resulting in increased yields [17,18]. In spinach, Rhizobium inoculation enhances vegetative growth, chlorophyll levels, and nitrogen content, while in lettuce and carrots, it increases dry matter production and phosphorus uptake [19]. Similarly, in corn, Rhizobium inoculation has been associated with increased plant height, leaf number, seed production, and forage yield [20].

Moreover, Gen-Jiménez et al. [21] demonstrated the effectiveness of native strains, including Rhizobium calliandrae, R. mayense, and R. jaguaris, in tomato crops. These strains promoted plant growth and fruit quality through mechanisms such as phosphate solubilization, siderophore production, indole acetic acid (IAA) synthesis, cellulose production, biofilm formation, and root colonization. However, studies on the use of Sinorhizobium strains as PPB are still very limited, and research focused on biofertilization using native rhizobial bacteria in Agave tequilana remains scarce. For this reason, investigations into the use of rhizobial bacteria as PPB in agave species are of great importance, as they provide a broad perspective on the implementation of sustainable agricultural biotechnologies for the cultivation of agro-industrially significant crops, such as A. tequilana.

Given the broad applications of A. tequilana in industries from alcoholic beverages and functional foods to medicinal products, biofertilization with PPB such as Rhizobium sp. ACO-34A, Sinorhizobium mexicanum ITTG R7^T, and S. chiapasense ITTG S70^T represents an innovative, sustainable strategy for enhancing agave productivity [22]. The application of PPB strains as biofertilizers has the potential to reduce reliance on chemical fertilizers and shorten the growth cycle of Agave tequilana by increasing the content of fermentable sugars such as inulin, sucrose, glucose, and fructose; this increase in sugar content is associated with enhanced plant growth and productivity, allowing the plants to reach maturity faster [23]. This approach supports the economic sustainability of agave cultivation while promoting environmentally friendly agricultural practices. This research focuses on evaluating the plant probiotic potential of native rhizobial strains in A. tequilana cultivation, specifically assessing their growth-promoting traits and their impact on sugar content.

2. Materials and Methods

2.1. Bacterial Strains

This study utilized the native rhizobial strain Rhizobium sp. ACO-34A (KM349967), isolated from the rhizosphere of Agave americana [24], along with Sinorhizobium mexicanum ITTG R7^T (DQ411930) and S. chiapasense ITTG S70^T (EU286550), both obtained from root nodules of the leguminous shrub Acaciella angustissima [25]. These bacterial strains are non-pathogenic and non-toxic to humans, animals, and the environment. All strains were sourced from the bacterial collection at the Laboratory of Genomic Ecology, Center for Genomic Sciences, UNAM (Morelos, Mexico).

2.2. Functional Analysis and Gene Annotation of PPB Traits in Native Rhizobial Strains

The functional analysis of plant probiotic bacteria (PPB) traits in the genomes of these strains was performed using the RAST-NMPDR web server [26,27]. Biosynthetic gene clusters associated with PPB potential were identified in the complete genomes using antiSMASH v.7.0.1 [28]. Additionally, genes related to nitrogen fixation, phosphate solubilization, siderophore production, biofilm formation, colonization potential, and tolerance to salinity and heavy metals were annotated using the PROKKA bioinformatics tool (http://vicbioinformatics.com/ (accessed on 11 May 2024)). The whole-genome nucleotide sequence of Rhizobium sp. ACO-34A has been deposited in GenBank under accession numbers CP021371 to CP021375 [24]. Likewise, the complete genome sequence of S. mexicanum ITTG R7^T is available in GenBank under accession numbers CP041238 to CP041241, along with SRA numbers SRR13240057 and SRR13240058 [29]. For S. chiapasense ITTG S70^T, the genome sequence is deposited under GenBank accession numbers CP133148 to CP133152, with the SRA project number PRJNA1007734.

2.3. Phenotypic Characterization of Rhizobial Strains

The morphological, physiological, and genomic characteristics of the strains ACO-34A, ITTG R7^T, and ITTG S70^T were thoroughly analyzed. Bacterial cell shape, size, and flagellar presence were examined using light microscopy, while Gram staining was performed following the manufacturer’s protocol (Merck^®, Darmstadt, Germany). Colony morphology was characterized in detail using PY medium according to established methods [25]. Salt tolerance was assessed at 28 °C across a range of NaCl concentrations, along with pH tolerance evaluations under varying conditions. Additionally, heavy metal tolerance was tested for aluminum (Al³⁺), copper (Cu²⁺), zinc (Zn²⁺), and lead (Pb²⁺) by culturing the rhizobial strains on PY medium supplemented with varying concentrations of 500 μM Al³⁺ and 100 μM for Cu²⁺, Zn²⁺, and Pb²⁺. The minimum inhibitory concentrations (MIC) for each metal were determined following the protocol described by Ajayi and Adekanmbi [30].

Intrinsic antibiotic resistance was evaluated using the disk diffusion method on PY-Ca²⁺ medium. Strains were evenly inoculated, and commercial antibiotic disks PT-36 Multibac^® (JAFS, Sinaloa, Mexico) targeting Gram-positive and Gram-negative bacteria were applied. The antibiotics tested included Netilmicin (10 mg), Penicillin (10 mg), Chloramphenicol (30 mg), Gentamicin (10 mg), Ciprofloxacin (5 mg), Cefalexin (30 mg), Amikacin (30 mg), and Ampicillin (10 mg). Plates were incubated at 28 °C for 48 h, and resistance (+) or susceptibility (−) was determined based on the presence or absence of inhibition zones [17].

2.4. Assessment of Plant Probiotic Traits in Rhizobial Strains

2.4.1. Phosphate Solubilization

The rhizobial strains were cultured individually in YM broth supplemented with 0.2% dicalcium phosphate (CaHPO₄) or tricalcium phosphate (Ca₃(PO₄)₂) as phosphate sources, with the pH adjusted to 7.0. After inoculation, plates were incubated at 28 °C for 15 days, during which phosphate solubilization was detected by the formation of halos around the rhizobial colonies. The Phosphate Solubilization Index (PSI) was calculated as the ratio of the halo diameter to colony diameter [31]. The concentration of soluble phosphorus was then determined using the ammonium vanadate-molybdate method, with absorbance measured at 420 nm on a UV/visible spectrophotometer, as outlined by O’Halloran & Cade-Menun [32]. In addition, rhizobial strains were cultured in 125 mL conical flasks containing 50 mL of liquid National Botanical Research Institute’s Phosphate (NBRIP) medium, composed of glucose (10 g), Ca₃(PO₄)₂ (5 g), MgCl₂·6H₂O (5 g), MgSO₄·7H₂O (0.25 g), KCl (0.2 g), and (NH₄)₂SO₄ (0.1 g) per liter at pH 7.0. Each flask was inoculated with 1 mL of a rhizobial suspension (1 × 10⁶ CFU mL⁻¹) and incubated at 30 °C with shaking at 125 rpm. Soluble phosphate concentrations were measured every 24 h over a period of 6 days, with uninoculated NBRIP medium serving as a control. All experiments were conducted in triplicate.

2.4.2. Siderophore Production

Siderophore production was assessed both qualitatively and quantitatively using the Chrome Azurol S (CAS) assay [33]. Rhizobial strains were streaked onto CAS-agar medium, containing CAS, ferric iron (Fe³⁺), and hexadecyltrimethylammonium bromide (HDTMA). After incubation at 28 °C for 3 days, colonies that formed an orange halo were classified as siderophore producers, and halo diameters were recorded. Siderophore production was expressed as the Siderophore-Induced Droplet (SID) index. For quantitative measurement, rhizobial strains in the exponential growth phase were cultured in 25 mL of King’s B broth in 125 mL flasks at 28 °C for 3 days. After centrifugation at 3000 rpm, 0.5 mL of the supernatant was mixed with 0.5 mL of CAS solution and 10 μL of sulfosalicylic acid. Following a 20-min incubation, absorbance was measured at 630 nm on a Beckman Coulter^® DU730 spectrophotometer, using uninoculated King’s B broth as a control. Siderophore production was calculated as follows: (Ar − As)/Ar × 100, where Ar is the absorbance of the reference and As is the absorbance of the sample [34].

2.4.3. Indole Acetic Acid (IAA) Production

IAA production was quantified through a colorimetric assay using Salkowski’s reagent, made by mixing 50 mL of 35% perchloric acid (HClO₄) with 1 mL of 0.5 M ferric chloride (FeCl₃), following the method by Bric et al. [35]. Each rhizobial strain was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of YMB medium supplemented with 2 g L⁻¹ L-tryptophan (medium composition: yeast extract, 5 g; mannitol, 10 g; KH₂PO₄, 0.2 g; K₂HPO₄, 0.1 g; MgCl₂, 0.2 g; MgSO₄·7H₂O, 0.2 g; CaCl₂, 0.02 g; and phenol red, 0.01 g per liter, pH 7.0). Cultures were incubated at 28 ± 2 °C for 7 days at 150 rpm. After centrifugation at 10,000 rpm for 10 min at 4 °C, the supernatant was mixed with Salkowski’s reagent in a 1:2 ratio and incubated in the dark at 28 ± 2 °C for 30 min. Absorbance was measured at 530 nm, and IAA concentrations were calculated using a standard curve. All measurements were conducted in triplicate.

2.4.4. Cellulose Production

Cellulose production by the native rhizobial strains was tested on Yeast Extract Mannitol (YEM) agar supplemented with 0.25% Congo Red. This medium was composed of mannitol (10 g), K₂HPO₄ (0.5 g), MgSO₄ (0.2 g), NaCl (0.1 g), CaCO₃ (3.0 g), and yeast extract (3.0 g) per liter, adjusted to pH 6.8. The Congo Red dye binds to β-1, 4 linkages characteristic of cellulose-like polysaccharides, enabling visual detection of cellulose production. Plates were incubated at 28 °C for 5 days, and cellulose-producing colonies were observed under a Zeiss^® Stemi 2000-C Stereo Microscope [36].

2.4.5. Cellulase Activity

Cellulase activity was determined on plates containing YEM agar supplemented with 1% carboxymethylcellulose (CMC), following the method by Hankin and Anagnostakis [37]. After 7 days of incubation at 28 °C, rhizobial colonies were removed with distilled water, and plates were stained with 0.1% Congo Red solution for 30 min. The plates were then washed with 1 M NaCl to reveal lysis halos, which indicated cellulase production [38].

2.5. Assessment of Biofilm Formation Ability

The biofilm-forming capacity of native rhizobial strains was quantitatively assessed using a 96-well flat-bottom polystyrene microtiter plate (Costar^®, Corning Inc., New York, NY, USA) and crystal violet staining [39]. Strains were cultured in PY-Ca²⁺ medium at 28 °C until reaching an optical density (OD₆₀₀) of 0.2 (approximately 1 × 10⁸ CFU mL⁻¹). Aliquots of 200 μL were inoculated into individual wells containing minimal medium and incubated statically at 28 °C for 72 h. After incubation, unbound cells and growth media were carefully removed, and wells were rinsed three times with sterile phosphate-buffered saline (PBS, pH 7.4) to eliminate loosely adherent cells. The biofilms were then stained with 200 μL of 0.3% (w/v) crystal violet solution (Sigma-Aldrich^®, St. Louis, MI, USA) for 10 min at room temperature. Excess stain was removed by rinsing the wells three times with deionized water, followed by air-drying for 15 min. The adhered crystal violet was solubilized with 200 μL of an 80% ethanol–20% acetone solution, and absorbance was measured at 570 nm using a microplate reader (Bio-Rad^® Model 680, Hercules, CA, USA) [16]. Biofilm production was expressed as relative absorbance units, normalized to account for variability in the initial inoculum. Statistical analysis of biofilm formation across strains was conducted using one-way ANOVA, followed by Fisher’s post hoc test (p < 0.05) in StatView 5.0 software (Abacus Corporation, Encino, CA, USA).

This standardized approach ensured reproducibility and accuracy in evaluating the biofilm-forming capabilities of the rhizobial strains.

2.6. Root Colonization Capacity Assay

To evaluate root colonization by rhizobial strains using scanning electron microscopy (SEM), Agave tequilana seeds were sterilized by immersion in 70% ethanol for 30 s, followed by 5 min in 5% sodium hypochlorite, and rinsed thoroughly with sterile distilled water. Sterilized seeds were germinated on water-agar plates, and 25 healthy seedlings were selected after 3 days of incubation in darkness [40]. Seedlings were inoculated with rhizobial suspensions (1 × 10⁸ CFU mL⁻¹) cultured in YMA medium at 28 °C for 48 h. Each seedling received 250 µL of bacterial suspension at the root-cotyledon junction and was incubated under controlled conditions for 15 days. For SEM, roots were washed with sterile distilled water to remove non-adherent bacteria and fixed in 2.5% glutaraldehyde (pH 7.2) for 12 h at 4 °C. Fixed samples were washed in phosphate buffer, dehydrated through a graded ethanol series, and critical-point dried using CO₂. Samples were mounted on stubs with carbon adhesive tape and coated with gold using a sputter coater. Imaging was performed with a high-resolution SEM under an accelerating voltage of 5–15 kV to observe bacterial adherence, biofilm formation, and colonization patterns on root surfaces. Images were captured to analyze biofilm structure and spatial distribution [41].

2.7. Plant Inoculation Assay in Agave tequilana

The effect of inoculation on the growth and sugar content of Agave tequilana plants was evaluated using five treatments: T₁ = Rhizobium sp. ACO-34A, T₂ = Sinorhizobium mexicanum ITTG R7^T, T₃ = Sinorhizobium chiapasense ITTG S70^T, T₄ = chemical fertilizer (17N-17P-17K), and T₅ = negative control (no inoculation, no fertilization). Plants at the ’piña’ stage were transplanted into pots containing a 2:1 sterilized mixture of Peat-Moss and perlite and inoculated with 10 mL of a bacterial suspension (10⁶ CFU mL⁻¹). The experimental design was completely randomized with four replicates per treatment, and plants were grown under greenhouse conditions for 90 days. Variables measured included total height, total fresh weight, piña weight, number of leaves, and carbohydrate content, with data recorded at the time of transplantation and after 90 days of growth. Carbohydrate content was quantified using high-performance liquid chromatography with infrared detection (HPLC-IR), employing inulin, fructose, sucrose, and glucose as standards. Samples were adjusted to pH 7.0 and filtered through 20- and 45-μm membranes before being placed in 2-mL vials. A 10-μL sample of plant extract was injected into an HPLC-IR (Thermo Finnigan^®, Ontario, Canada) equipped with a Rezex RCM-monosaccharide Ca²⁺ column, with water as the mobile phase at a temperature of 85 °C, a flow rate of 0.3 mL min⁻¹, and a pressure of 300 psi [42]. Data were analyzed using analysis of variance (ANOVA), followed by post-hoc comparisons with Tukey’s test (p < 0.05).

3. Results

3.1. Biosynthetic Gene Clusters in Native Rhizobial Strains with PGP Potential

The genomic analysis of Rhizobium sp. ACO-34A, S. mexicanum ITTG R7^T, and S. chiapasense ITTG S70^T highlights their plant probiotic traits, reinforcing their potential as biofertilizers for sustainable agriculture (Figure 1). Rhizobium sp. ACO-34A (Figure 1a) possesses a 6.28 Mb genome with 61% GC content, comprising one chromosome (4.75 Mb) and four extrachromosomal replicons: two plasmids (516 kb and 213 kb) and two chromids (494 kb and 305 kb). Its genome includes genes associated with nitrogen fixation (nifHDK operon), phosphate solubilization, siderophore production, biofilm formation, and cellulose synthesis. The 516-kb plasmid harbors a ribosomal operon, while the truncated symbiosis plasmid (213 kb) retains nitrogen fixation genes, such as nifH, but lacks nod genes required for legume symbiosis.

S. mexicanum ITTG R7^T (Figure 1b), with a 7.14 Mb genome and GC content of 59–62%, consists of one chromosome (4.31 Mb) and three plasmids (436 kb, 455 kb, and 1933 kb). Its 455-kb plasmid (pSym) harbors nod and nif genes critical for nitrogen fixation, including additional nifX and nifN genes for FeMo-Co biosynthesis. The 1933-kb plasmid is rich in carbohydrate transport and metabolism genes, while the genome also contains secretion system genes (types I-IV) and multiple phn clusters, enhancing adaptability to diverse phosphorus sources.

S. chiapasense ITTG S70^T (Figure 1c), with a genome of one chromosome (4.3 Mb) and four plasmids (1600 kb, 442 kb, 279 kb, and 174 kb) and GC content of 58.61–61.85%, is notable for its multidrug resistance genes (MdtA-N), which may confer a competitive advantage in the rhizosphere. These MDR genes are primarily categorized under the “Stress Response” subsystem, where they contribute to resistance against environmental and chemical stressors, ensuring the survival and competitive fitness of the bacteria.

Subsystem analysis across all strains reveals significant genes related to stress response, metabolism, and membrane transport, supporting resilience to salinity, pH, and heavy metals. These strains collectively exhibit traits such as phosphate solubilization, siderophore production, auxin synthesis, biofilm formation, and cellulose production, emphasizing their adaptability and biofertilizer potential for enhancing plant health and resilience in diverse agricultural environments. Genes associated with biofilm formation are primarily categorized under the “Cell Wall and Capsule” subsystem. This includes genes involved in the biosynthesis of exopolysaccharides, which are essential for forming the extracellular matrix of biofilms, and cellulose production (e.g., bcsA, bcsB, and bcsC), as well as other structural components necessary for biofilm development. For Rhizobium sp. ACO-34A, this subsystem is strongly represented, correlating with its robust biofilm production. Similarly, S. mexicanum ITTG R7^T and S. chiapasense ITTG S70^T harbor biofilm-related genes within this subsystem, albeit with variations that explain differences in biofilm formation.

In addition, the “Regulation and Cell Signaling” subsystem includes regulatory genes such as expR and luxR, which govern exopolysaccharide production and quorum sensing pathways.

3.2. Phenotypic, Genomic, and Tolerance Characteristics of Rhizobial Strains

The results highlight significant physiological, morphological, and adaptive traits of the rhizobial strains Rhizobium sp. ACO-34A, S. mexicanum ITTG R7^T, and S. chiapasense ITTG S70^T, underscoring their potential as plant probiotic bacteria for Agave tequilana cultivation (

Table 1. Phenotypic characteristics of native rhizobial strains.

Characteristic	Rhizobium sp. ACO-34A	S. mexicanum ITTG R7T	S. chiapasense ITTG S70T
Origin	Agave americana (Mexico)	Acaciella angustissima (Mexico)	Acaciella angustissima (Mexico)
Cell morphology	Rods (0.4 × 1.1 μm)	Rods (0.7 × 1.2 μm)	Rods (0.6 × 1.4 μm)
Flagella	Peritrichous	peritrichous	peritrichous
DNA G+C content (mol%)	61.1	62.0	61.8
pH range for growth	5.0–8.0	5.0–8.0	5.0–8.0
Growth at/in:
37 °C	(−)	(−)	(−)
1% NaCl	(+)	(+)	(+)
2% NaCl	(+)	(+)	(+)
5% NaCl	(−)	(−)	(−)
Antibiotic resistance
(mg mL−1):
Netilmicin (10)	(−)	(+)	(+)
Penicillin (10)	(+)	(+)	(+)
Chloramphenicol (30)	(+)	(+)	(+)
Gentamicin (10)	(+)	(−)	(−)
Ciprofloxacin (5)	(+)	(+)	(+)
Cefalexin (30)	(−)	(−)	(−)
Amikacin (30)	(−)	(+)	(−)
Ampicillin (10)	(+)	(+)	(+)
Tolerance to heavy metals (μM):
Al3+ (500 μM)	(+)	(+)	(+)
Cu2+ (100 μM)	(+)	(+)	(+)
Zn2+ (100 μM)	(−)	(+)	(+)
Pb2+ (100 μM)	(−)	(−)	(−)

(+), positive (resistance, growth observed); (−), negative (susceptibility, no growth observed).

). Morphological analysis revealed that all strains are rod-shaped, with dimensions of 0.4 × 1.1 µm for Rhizobium sp. ACO-34A, 0.7 × 1.2 µm for S. mexicanum ITTG R7 ^T, and 0.6 × 1.4 µm for S. chiapasense ITTG S70 ^T, and all possess peritrichous flagella, facilitating motility and root colonization. Genomic analysis indicated similar GC content across the strains: Rhizobium sp. ACO-34A (61.1%), S. mexicanum ITTG R7^T (62.0%), and S. chiapasense ITTG S70^T (61.8%), reflecting moderate genetic stability.

Physiological assessments demonstrated their moderate salinity tolerance, with all strains growing effectively at 1% and 2% NaCl but inhibited growth at 5% NaCl under laboratory conditions, indicating their potential for further evaluation in different soils under real field management. The antibiotic resistance profiles varied significantly, with ACO-34A showing resistance to penicillin, chloramphenicol, gentamicin, and ciprofloxacin, while ITTG R7^T and ITTG S70^T exhibited broader resistance, including to netilmicin and ampicillin. Regarding heavy metals, all strains tolerated aluminum (Al³⁺) and copper (Cu²⁺); however, only ITTG R7^T and ITTG S70^T were resilient to zinc (Zn²⁺), a trait advantageous in zinc-contaminated soils, while none tolerated lead (Pb²⁺). The concentrations of heavy metals used in this study (Al³⁺ 500 μM, Cu²⁺ 100 μM, Zn²⁺ 100 μM, and Pb²⁺ 100 μM) were selected based on previous studies evaluating rhizobial tolerance to environmental stressors. These concentrations represent levels commonly found in contaminated or naturally metal-rich soils, where rhizobia often coexist with other microbial communities. For example, Cu²⁺ and Zn²⁺ concentrations of 100 μM align with thresholds widely reported for evaluating microbial tolerance in agricultural and industrial contexts, while Al³⁺ at 500 μM reflects its solubility and bioavailability in acidic soils, typical of A. tequilana cultivation regions. Lead (Pb²⁺) at 100 μM corresponds to sublethal concentrations observed in anthropogenically impacted soils.

All strains thrived across a broad pH range (5.0–8.0) and demonstrated growth at temperatures up to 37 °C, supporting their application across diverse soil types and climates. These findings establish Rhizobium sp. ACO-34A as a promising biofertilizer for A. tequilana, with S. mexicanum ITTG R7^T and S. chiapasense ITTG S70^T offering additional benefits, particularly in zinc-enriched soils, contributing to sustainable agricultural practices under challenging environmental conditions.

3.3. Plant Probiotic (PPB) Potential of Native Rhizobial Strains

The results highlight the distinct plant probiotic capabilities of the three native rhizobial strains, emphasizing their strengths in phosphate solubilization, siderophore production, indole-3-acetic acid (IAA) production, nitrogenase activity, and cellulose-related abilities (

Table 2. Plant probiotic activity of native rhizobial strains.

Strain	P-Solubilization			Siderophore Production		IAA Production (mg L−1)
	PSI ¥	Ca3(PO4)2 (mg L−1)	CaHPO4 (mg L−1)	SID ≠	% Siderophore
Rhizobium sp. ACO 34A	1.17 ± (0.11) *	34.8 ± (0.65)	28.4 ± (0.76)	1.22 ± (0.06)	32.4 ± (0.84)	22.5 ± (0.65)
Sinorhizobium mexicanum ITTG R7	2.12 ± (0.18)	51.4 ± (1.39)	32.5 ± (0.93)	1.51 ± (0.08)	50.4 ± (0.83)	20.7 ± (0.57)
Sinorhizobium chiapasense ITTG S70	2.24 ± (0.21)	58.2 ± (1.27)	28.2 ± (0.87)	1.61 ± (0.07)	58.5 ± (0.92)	20.8 ± (0.66)

* Mean values of three replicates. The values in parentheses are standard deviations; PSI ^¥: Phosphate Solubilization Index; SID ^≠: Siderophore-Induced Droplet.

). These traits are crucial for promoting plant growth and enhancing soil nutrient availability, with each strain exhibiting unique advantages.

In phosphate solubilization, S. chiapasense ITTG S70^T demonstrates the highest efficiency, achieving a phosphate solubilization index (PSI) of 2.24 ± 0.21 and solubilization values of 58.2 ± 1.27 mg L⁻¹ for Ca₃(PO₄)₂ and 28.2 ± 0.87 mg L⁻¹ for CaHPO₄. This strain is closely followed by S. mexicanum ITTG R7^T, which exhibits a PSI of 2.12 ± 0.18 and solubilization values of 51.4 ± 1.39 mg L⁻¹ for Ca₃(PO₄)₂ and 32.5 ± 0.93 mg L⁻¹ for CaHPO₄. Rhizobium sp. ACO-34A, while slightly less effective, still shows moderate activity, with a PSI of 1.17 ± 0.11 and solubilization values of 34.8 ± 0.65 mg L⁻¹ for Ca₃(PO₄)₂ and 28.4 ± 0.76 mg L⁻¹ for CaHPO₄. These findings suggest that the ability to mobilize phosphorus varies significantly among the strains, with S. chiapasense ITTG S70^T being the most effective, making it particularly suitable for nutrient-poor soils.

Siderophore production follows a similar trend, further reinforcing the probiotic potential of these strains. S. chiapasense ITTG S70^T exhibits the highest siderophore activity, with a siderophore index (SID) of 1.61 ± 0.07 and production levels of 58.5% ± 0.92. S. mexicanum ITTG R7^T also shows strong siderophore production, with an SID of 1.51 ± 0.08 and 50.4% ± 0.83 production levels. Conversely, Rhizobium sp. ACO-34A demonstrates lower siderophore activity, with an SID of 1.22 ± 0.06 and 32.4% ± 0.84 production levels. These results indicate that while all three strains have the potential to enhance iron acquisition in plants, S. chiapasense ITTG S70^T is the most effective, making it particularly beneficial in iron-limited soils.

IAA production, a vital trait for promoting root elongation and development, is another area where S. chiapasense ITTG S70^T excels, producing the highest levels of IAA at 22.5 ± 0.65 mg L⁻¹. S. mexicanum ITTG R7^T follows with 20.7 ± 0.57 mg L⁻¹, while Rhizobium sp. ACO-34A produces 20.8 ± 0.66 mg L⁻¹. Although all strains demonstrate substantial auxin production, S. chiapasense ITTG S70^T maintains a consistent edge, further supporting its role as a superior plant growth promoter.

In terms of cellulose-related capabilities, Rhizobium sp. ACO-34A stands out. Congo red assays reveal that ACO-34A produces an intense reddish hue, indicating significant cellulose production (Supplementary Figure S1). In contrast, the colonies of ITTG R7^T and ITTG S70^T appear nearly white, suggesting minimal or no cellulose production in these strains. Enzymatic assays further highlight ACO-34A’s superiority in cellulase activity, followed by ITTG S70^T. Despite relatively weak lytic activity, ACO-34A’s ability to synthesize cellulose emphasizes its capacity to form exopolysaccharides, essential for biofilm production and strong plant-microbe interactions.

3.4. Biofilm Production by Native Rhizobial Strains

The biofilm-forming ability of the rhizobial strains was quantitatively evaluated by measuring absorbance at 570 nm at three time points (24, 48, and 72 h). Distinct biofilm production patterns were observed among the strains, indicating significant differences in their colonization capacities (Figure 2). The data presented in Figure 2 correspond to biofilm formation measured from undiluted bacterial suspensions, representing the maximum biofilm-forming capacity of each strain. While serial dilutions were used during protocol standardization, this figure specifically focuses on undiluted suspensions to provide clear insights into the strains’ full biofilm-forming potential.

At 24 h, all strains exhibited low biofilm production, with absorbance values ranging from 0.2 to 0.3. Rhizobium sp. ACO-34A recorded a slightly higher absorbance (0.3 ± 0.05) compared to S. chiapasense ITTG S70^T (0.25 ± 0.03) and S. mexicanum ITTG R7^T (0.2 ± 0.04), although the differences were not statistically significant.

At 48 h, a significant increase in biofilm production was observed for all strains (p < 0.05). ACO-34A demonstrated the highest biofilm production at this time point, with an absorbance value of 0.7 ± 0.1. Similarly, ITTG S70^T showed a marked increase to 0.6 ± 0.09, while ITTG R7^T exhibited a comparatively moderate increase to 0.5 ± 0.08. By 72 h, biofilm production further increased significantly (p < 0.05) across all strains. Rhizobium sp. ACO-34A maintained its lead with the highest absorbance value (0.9 ± 0.12), followed closely by ITTG S70^T (0.85 ± 0.11). S. mexicanum ITTG R7^T exhibited the lowest absorbance at this time point (0.8 ± 0.1). Statistical analysis revealed significant differences among the strains, with ACO-34A exhibiting significantly higher biofilm production than ITTG R7^T (p < 0.01), while the differences between ITTG S70^T and ACO-34A were less pronounced.

These results indicate that Rhizobium sp. ACO-34A exhibits the most robust biofilm formation dynamics, particularly after 48 h, likely due to its superior capacity for exopolysaccharide production. S. chiapasense ITTG S70^T also demonstrated strong biofilm production, following a similar trend but with slightly lower yields. In contrast, ITTG R7^T displayed a more linear increase in biofilm formation, with consistently lower production compared to the other two strains. These differences may be attributed to variations in the production and composition of exopolysaccharides, which influence the structural and functional characteristics of the biofilms formed by each strain.

3.5. Colonization Capacity of Native Rhizobial Strains

Using scanning electron microscopy (SEM), the colonization patterns of native rhizobial strains on Agave tequilana roots were observed. SEM analysis demonstrated the strains ability to adhere to root surfaces, with distinct colonization intensities and detailed visualizations of bacterial attachment (Figure 3).

Rhizobium sp. ACO-34A exhibited strong colonization as early as 5 days post-inoculation (dpi), with scattered bacterial clusters on the basal regions of secondary roots. By 7 dpi, colonization became more organized, forming dense accumulations along root grooves. At 12 and 15 dpi, a geometric pattern of extensive bacterial coverage was evident. SEM confirmed these findings, showing numerous rod-shaped bacteria tightly adhering to the root surface, often surrounded by visible exopolysaccharide layers (Figure S1).

S. mexicanum ITTG R7^T displayed moderate colonization, with bacteria adhering primarily to root hairs and forming dispersed clusters by 7 dpi. Over time, colonization extended along the root surface, forming localized patches that followed the alignment of epidermal cells. SEM images revealed bacteria concentrated at intercellular junctions, although the production of exopolysaccharides was less prominent compared to ACO-34A.

Sinorhizobium chiapasense ITTG S70^T exhibited diffuse colonization during the initial 5 dpi, with bacteria primarily localized at the apical ends of roots by 7 dpi. At 12 and 15 dpi, colonization became more uniform but was less intense than that observed for the other strains. SEM imaging displayed scattered bacterial distribution on the root surface, with minimal clustering and weak biofilm formation.

These results underscore the superior colonization and biofilm-forming capacity of ACO-34A, followed by ITTG R7^T, while ITTG S70^T exhibited the weakest colonization. Variability in exopolysaccharide production likely contributed to these differences in colonization patterns.

3.6. Growth Parameters of Agave tequilana Inoculated with Plant Probiotic Bacteria

The morphometric analysis of A. tequilana plants inoculated with native rhizobial strains revealed significant differences across treatments (p < 0.05) in all evaluated parameters (

Table 3. Morphometric parameters of Agave tequilana plants inoculated with native rhizobial strains.

Treatments	Total Height (cm)	Total Fresh Weight (g)	Piña Weight (g)	Number of Leaves	Chlorophyll (mg/g FW)
ACO-34A	77.20 (±8.79) *A	1416.80 (±181.33) A	852.45 (±154.67) A	14.25 (±0.96) A	861.12 (±46.27) A
ITTG R7T	50.95 (±6.52) C	835.70 (±152.47) B	637.87 (±90.13) B	13.75 (±1.03) B	758.82 (±17.40) B
ITTG S70T	52.97 (±5.60) C	801.97 (±176.71) B	370.70 (±119.27) C	11.00 (±0.5) C	629.97 (±57.80) C
Chemical fertilizer (Triple 17)	57.21 (±4.12) B	1431.71 (±191.53) A	604.50 (±62.35) B	10.52 (±0.5) C	649.9 (±35.67) C
Negative control	40.30 (±10.00) D	586.12 (±190.21) C	280.27 (±159.85) D	8.00 (±1.34) D	664.15 (±43.09) C
p-value	0.0000	0.0000	0.0000	0.0000	0.0000
HSD Tukey ¥ (p < 0.05)	3.9804	90.2026	47.062	2.2334	50.2347

* Mean values of four replicates. Means followed by the same letter are non-significant (Tukey test, p < 0.05);. ^¥ HSD: Honest Significant Difference.

). Plants treated with ACO-34A consistently demonstrated superior growth performance compared to other treatments, achieving the highest total height (77.20 cm), total fresh weight (1416.80 g), and chlorophyll content (861.12 mg/g FW). These values were statistically significant according to Tukey’s test.

In terms of piña weight, ACO-34A inoculated plants also ranked highest (852.45 g), significantly different from plants treated with chemical fertilizer (604.50 g). The chemical fertilizer treatment produced comparable results to ACO-34A in total fresh weight (1431.71 g) but was less effective in enhancing plant height and chlorophyll content.

ITTG R7^T and S. chiapasense ITTG S70^T showed moderate performance, with total fresh weight values of 835.70 g and 801.97 g, respectively. However, these treatments resulted in lower piña weights and reduced chlorophyll content, with significant differences (p < 0.05) compared to the top-performing treatments.

The negative control (no inoculation, no fertilization) consistently underperformed, recording the lowest values across all parameters, including total height (40.30 cm), fresh weight (586.12 g), and piña weight (280.27 g). These results highlight the crucial role of rhizobial inoculation in enhancing A. tequilana growth under controlled conditions.

Collectively, the findings underscore the efficacy of ACO-34A as a biofertilizer, with its performance similar to that of chemical fertilizer, thus demonstrating its potential as a sustainable alternative for improving growth parameters in A. tequilana.

3.7. Effect of Biofertilization on Sugar Content in Agave tequilana Plants

The sugar content in Agave tequilana plants inoculated with native rhizobial strains showed statistically significant differences (p < 0.05) across the treatments (

Table 4. Sugar content in Agave tequilana plants inoculated with native rhizobial strains.

Treatments	Inulin (mg g−1)	Sucrose (mg g−1)	Glucose (mg g−1)	Fructose (mg g−1)
ACO-34A	4.03 (±0.15) A *	2.77 (±0.08) A	3.07 (±0.18) A	2.49 (0.09) A
ITTG R7T	2.29 (±0.13) B	1.70 (±0.06) B	2.22 (±0.06) B	2.16 (0.09) A
ITTG S70T	2.15 (±0.13) B	1.66 (±0.07) B	2.19 (±0.11) B	2.14 (0.14) A
Chemical fertilizer (Triple 17)	2.24 (±0.11) B	1.68 (±0.10) B	2.32 (±0.06) B	2.29 (0.13) A
Negative control	1.13 (±0.02) C	1.17 (±0.04) C	1.6 (±0.03) C	1.25 (0.06) B
p-value	0.0000	0.0003	0.0005	0.0065
HSD ¥ (p < 0.05)	0.3485	0.6425	0.6204	0.7988

* Mean values of four replicates. Means followed by the same letter are non-significant (Tukey test, p < 0.05); ^¥ HSD: Honest Significant Difference.

). Plants inoculated with Rhizobium sp. ACO-34A exhibited the highest concentrations of inulin, sucrose, glucose, and fructose compared to all other treatments. Specifically, the inulin content in plants treated with ACO-34A (4.03 mg g⁻¹) was significantly higher than those treated with S. mexicanum ITTG R7^T (2.29 mg g⁻¹), S. chiapasense ITTG R7^T (2.15 mg g⁻¹), and chemical fertilizer (2.24 mg g⁻¹). The negative control had the lowest inulin content (1.13 mg g⁻¹), demonstrating its inferior performance. Similarly, sucrose levels followed the same trend, with ACO-34A achieving the highest sucrose concentration (2.77 mg g⁻¹), significantly surpassing those observed in the other inoculated treatments and the chemical fertilizer group, all of which ranged from 1.66–1.70 mg g⁻¹. The negative control showed the lowest sucrose concentration (1.17 mg g⁻¹).

For glucose, plants inoculated with ACO-34A again demonstrated superior levels (3.07 mg g⁻¹). The glucose levels in plants treated with other rhizobial strains and chemical fertilizer were comparable, while the negative control recorded significantly lower glucose. In terms of fructose, no significant differences were observed between treatments, except for the negative control, which exhibited significantly lower levels (1.25 mg g⁻¹).

4. Discussion

PPB are instrumental in advancing sustainable agriculture by enhancing crop productivity while reducing dependence on chemical inputs [43]. This study characterizes Rhizobium sp. ACO-34A, Sinorhizobium mexicanum ITTG R7^T, and S. chiapasense ITTG S70^T, underscoring their potential as biofertilizers for Agave tequilana Weber var. Blue. Genomic analysis of ACO-34A revealed a genome size of 6.28 Mb with a GC content of 61%, organized into a chromosome and four extrachromosomal replicons. This strain possesses genes linked to phosphate solubilization, siderophore production, and biofilm formation, which collectively enhance its adaptability and functional versatility [24]. Unlike canonical rhizobia, ACO-34A lacks nod genes yet retains a truncated symbiosis plasmid (213 kb), suggesting evolutionary adaptation for interaction with non-leguminous hosts, similar to traits reported for Rhizobium laguerreae in non-legume crops [19]. Comparatively, S. mexicanum ITTG R7^T and S. chiapasense ITTG S70^T harbor larger plasmids enriched with multidrug resistance genes and phosphorus metabolism pathways, highlighting their competitiveness in diverse rhizospheric environments [25].

Phenotypic analyses demonstrated that ACO-34A exhibits notable resilience under abiotic stress, including tolerance to a broad pH range (5.0–8.0), temperatures up to 37 °C, and salinity levels of up to 2% NaCl. Its ability to tolerate aluminum and copper, combined with selective antibiotic resistance, aligns with its genomic repertoire of stress-response genes, making it highly adaptable to challenging agricultural soils. Additionally, ACO-34A effectively colonizes roots and forms robust biofilms, critical for establishing plant-microbe interactions. While ITTG R7^T and ITTG S70^T exhibit broader antibiotic resistance and greater zinc tolerance, ACO-34A stands out for its consistent performance under multiple stressors, reinforcing its functional versatility as a biofertilizer [44].

The probiotic traits of ACO-34A further enhance its biofertilizer potential. It exhibits moderate phosphate solubilization, particularly in dicalcium and tricalcium phosphate forms, improving phosphorus availability in nutrient-deficient soils. This process, facilitated by the secretion of organic acids such as gluconic, acetic, and succinic acids, mirrors mechanisms documented in other rhizobial strains such as Rhizobium calliandrae and R. jaguaris [21,45]. Additionally, ACO-34A produces siderophores—iron-chelating molecules that improve iron bioavailability—essential for plant enzymatic processes and overall growth [46]. These traits have also been linked to the suppression of phytopathogenic fungi, providing the dual benefits of nutrient acquisition and disease mitigation [47,48].

The synthesis of indole-3-acetic acid (IAA), a key auxin promoting root elongation and development, further distinguishes ACO-34A as a plant probiotic bacterium. Like other Rhizobium species, such as R. laguerreae, which enhances root systems in spinach and lettuce [19], ACO-34A demonstrates substantial IAA production, fostering robust root architecture. Additionally, its superior cellulose synthesis and cellulase activity, as confirmed through Congo red assays, underpin its biofilm-forming capacity and its ability to colonize root surfaces effectively. These findings align with reports on cellulase activity in Phyllobacterium sp. PEPV15, which facilitates root colonization and nutrient exchange in strawberries [16].

Scanning electron microscopy revealed dense bacterial clusters along A. tequilana roots, confirming robust colonization. This trait is critical for nutrient exchange, competitive exclusion of other microorganisms, and is essential for maintaining structural integrity and functional efficacy under environmental stress [49,50]. In rhizospheric environments, biofilm-forming bacteria provide spatial organization and multiple levels of protection, enhancing microbial resilience and functionality [51,52].

In greenhouse inoculation assays, ACO-34A consistently outperformed other treatments, significantly enhancing plant height, fresh weight, and chlorophyll content. These effects often surpassed those achieved with chemical fertilizer, underscoring its potential as a sustainable alternative. Enhanced sugar content, particularly inulin and fructose, further emphasizes its agro-industrial relevance. Fructan synthesis, driven by enzymatic pathways involving fructosyltransferase and sucrose synthase, positions ACO-34A as a valuable biofertilizer for prebiotic and nutraceutical applications [53]. Comparisons with other studies, such as increased phenolic and chlorophyll content in spinach and lettuce inoculated with R. laguerreae, reinforce the multifunctional role of native rhizobia as plant probiotics in enhancing crop quality and resilience [13]. These results highlight Rhizobium sp. ACO-34A as a promising candidate for sustainable Agave tequilana production, demonstrating its ability to improve plant growth while enhancing the nutritional and industrial value of the crop.

5. Conclusions

This study evaluated the plant probiotic potential of Rhizobium sp. ACO-34A, Sinorhizobium mexicanum ITTG R7^T, and S. chiapasense ITTG S70^T for enhancing growth and sugar content in Agave tequilana. The native rhizobial strains demonstrated a versatile genomic profile, including genes linked to phosphate solubilization, siderophore production, and biofilm formation, highlighting their adaptability to diverse environmental conditions. Phenotypic assessments revealed notable resilience to salinity, pH variability, and metal tolerance, along with superior root colonization and biofilm-forming capacities. Key probiotic traits, such as efficient phosphate solubilization, IAA synthesis, and cellulose production, further underscored their biofertilizer potential. Inoculation with Rhizobium sp. ACO-34A significantly improved plant growth parameters and sugar content, particularly inulin and fructose levels, demonstrating its relevance for agro-industrial and nutraceutical applications. Among the tested strains, ACO-34A demonstrated superior performance, establishing itself as a promising biofertilizer candidate. Its versatile genomic profile includes genes linked to phosphate solubilization, siderophore production, and biofilm formation, which likely contribute to its adaptability to diverse environmental conditions. These findings position strain ACO-34A as a sustainable alternative to chemical fertilizers, offering significant potential for broader application in other high-value crops to promote sustainable agricultural practices.