Millet is a small seeded cereal crop from the family Poaceae, classified under the C4 grasses, and usually cultivated in xeric and semi-xeric regions (Han et al., 2017). Major and minor millets are two categories of millets which include Eleusine coracan (finger millet), Panicum miliaceum (proso millet), Setaria italica (foxtail millet), Pennisetum glaucum (pearl millet), Echinochlo acolona (barnyard millet), Digitaria bursa (black fonio millet), Digitaria exilis (white fonio millet), Panicum miliare (little millet), Paspalum scrobiculatum (Kodo millet), Eragrostis tef (teff millet), respectively (Annor et al., 2014). Millets are known for their potential to withstand harsh, dry environments and challenging environments, including changing climates and nutrient-poor soil (Sharma and Ortiz, 2000). These characteristic features make it a wise alternative cereal crop for regions with limited rainfall, irrigation, and nutrient-poor soil. Millets typically flourish in higher temperatures and can reproduce with minimal water input, making them thermophilic and xerophilic (Saxena et al., 2018). Together with their nutritional benefits, millets offer short-term cultivation periods, reduced water requirements, and adaptability to adverse climatic conditions. When compared to major cereal crops, millets yield high on marginal land. These specific agro-climatic characteristics render millets particularly well-suited for cultivation in semi-arid regions of Asia and Africa, where other major crops frequently encounter challenges (Belton and Taylor, 2004).

The rhizosphere is an area of the soil that harbors microorganisms with a significant connection to plant physiology, involving complex organic material decomposition, resistance to stresses (both biotic and abiotic), and contributing to plant development. The resilience of millets is largely driven by the microbial community present in the rhizosphere. The term “plant growth-promoting microorganisms” (PGPM) encompasses a diverse array of organisms that reside in the rhizosphere, which includes plant growth-promoting rhizobacteria (PGPR), antagonistic microorganisms, beneficial symbionts, mycorrhizal fungi, mycoparasitic fungi, endophytic bacteria, and fungi (Meena et al., 2017). The PGPR present in the rhizosphere region boosts plant growth and development by engaging in various positive mechanisms. These include increasing the nutrient uptake, fixing the atmospheric nitrogen, suppressing the growth of phytopathogens (Wang et al., 2022; Sheteiwy et al., 2023), and improving the host plant resistance against various abiotic and biotic stressors (Selvakumar et al., 2012; Zolla et al., 2013) and results in increased plant growth and yield (Berg, 2009; Huang et al., 2014).

The principal role of PGPR in nurturing plant growth is achieved through two distinct mechanisms: direct and indirect (Kong and Liu, 2022). The direct mechanisms involve the synthesis of plant growth hormones such as cytokinin, gibberellins, and auxins, together with the solubilization of nutrients, including phosphate. The indirect mechanisms include the production of antibiosis to combat phytopathogens and ACC deaminase to resist various stressors (Barea et al., 2005). Azotobacter, Azospirillum, Acinetobacter, Azoarcus, Arthrobacter, Burkholderia, Bacillus, Beijerinckia, Enterobacter, Erwinia, Klebsiella, Gluconacetobacter, and Serratia. Some of the most extensively studied PGPR for plant growth promotion (PGP) belong to the genus Rhizobium and Pseudomonas (Murphy et al., 2003; Esitken et al., 2006). Acinetobacteria have been shown to enhance the yields of various crops, particularly those of global importance like rice, maize, and wheat (Mahmoud et al., 2024).

To address the growing food demands of an increasing population, agrochemicals use has escalated to boost crop growth and productivity. These agrochemicals are pivotal in increasing crop productivity. However, their prolonged usage may lead to soil infertility through processes such as diminishment of soil organic matter, nitrogen leaching, depletion of soil carbon, and soil compaction. To overcome the usage of hazardous chemicals in agriculture, a wise alternative is the application of biofertilizers derived from PGPR (Nader et al., 2024), while endophytes (Mahmoud et al., 2025) can also act as promising agents that enhance plant growth and support eco-friendly, sustainable agriculture. It is an environmentally friendly and cost-effective alternative that can enhance soil fertility and pave the way for sustainable agriculture. This research work intends to isolate, characterize, and identify PGPRs that possess PGP properties from the region’s millet rhizosphere.

In the rhizosphere, PGPRs reside on the root surface, promoting plant growth and development through both direct and indirect mechanisms (Meena et al., 2017). These mechanisms include nitrogen fixation, growth hormone production (auxins, gibberellins, cytokinins, etc.), ammonia production, inorganic phosphate solubilization, and the suppression of the growth of antagonistic microorganisms (Gerhardt et al., 2009). In the present work, 53 morphologically distinct isolates were isolated from millet rhizosphere soil using the spread plate technique. All the isolates were assessed for their nitrogen fixation ability, and 26 were found to be capable of fixing nitrogen when inoculated in a nitrogen-free medium. Nitrogen is a crucial element for the growth of the plant and yield; however, plants are unable to use nitrogen directly. The advantageous microorganisms found in the rhizosphere region are capable of converting atmospheric nitrogen into a usable form, namely ammonia, which is a major component of nucleic acids, amino acids, chlorophyll, and ATP (Andy et al., 2020). Similar results were reported by Reang et al. (2022) and Kanimozhi and Panneerselvam (2010) using the same medium, demonstrated that the isolates belonging to the genus Halomonas and Azospirillum, respectively, possess the ability to fix soil atmospheric nitrogen

It has been established that the synthesis of auxin by PGP bacteria (PGPB) represents the primary mechanism through which these bacteria can improve the growth of plants. In particular, the majority of the scientific literature focuses on IAA, the most prevalent biologically active auxin, which plays a pivotal role in plant growth by enhancing root and shoot development, elongation of roots and stems differentiation of vascular tissues, cell expansion, loosening of root cell walls, root bacterial colonization, stimulation of cell division, and defense against pathogens (Orozco-Mosqueda et al., 2023). In the present study, 14 of the 26 bacterial isolates produced IAA at varying concentrations; the highest IAA production of 0.37mg/mL was observed in SA1, and the lowest was in R5 (0.05 mg/mL). A comparable outcome was noted in the research conducted by the authors (Walpola and Arunakumara, 2016), they documented the maximum IAA production of 240 µg/mL and 332 µg/mL by Enterobacter ludwigii and Enterobacter hormaechei, respectively.

Ammonia production is a significant factor associated with the PGPB. Plants utilize the released ammonia from the PGPB as a nitrogen source, and the soil with abundant ammonia becomes alkaline, which prevents the growth of some fungi (Jha et al., 2012). In the current work, 23 bacterial isolates demonstrated positive results for ammonia production. Several reports exist on the production of ammonia in various concentrations by the genus Bacillus, particularly B. licheniformis, reported to produce nearly 4 µmol/mL (Goswami et al., 2014).

Phosphorus is a pivotal element that holds a crucial role in plant nutrition and numerous metabolic functions, including photosynthesis, respiration, energy transmission, signal transduction, and the biosynthesis of macromolecules (Anand et al., 2016). The majority of phosphorus, typically ranging from 95 to 99%, is present in an insoluble form that is not directly usable by plants. This can be achieved through the use of PGPB, which can lower the rhizosphere pH by secreting organic acids, including carboxylic acid and succinic acid. These acids bind to the phosphate group, forming calcium phosphate, thereby converting the insoluble phosphate to an available form (Sharma et al., 2013). In the present work, 15 isolates exhibited positive results with varying PSI values ranging from 2.36 to 3.80. The highest PSI was observed in P. agglomerans with a value of 3.80, and a similar result, with a 0.3 PSI difference, was reported in the research conducted by Prasad et al. (2022).

HCN is a volatile compound, a secondary metabolite, that inhibits the growth of phytopathogens (Kumar et al., 2014). Additionally, HCN production plays a pivotal role in root and shoot elongation, biomass production, and nitrogen accumulation (Marques et al., 2010). In the present work, only three isolates, namely R4 (E. hormaechei), R5 (B. siamensis), and MS3 (B. velezensis), demonstrated the capacity to produce HCN. Katiyar et al. (2017) documented the production of HCN by E. hormaechei, while Kanchiswamy et al. (2015) noted the production of HCN by the genus Bacillus.

The application of PGPB with siderophore-producing potential has been demonstrated to enhance plant growth by boosting iron intake, while simultaneously inhibiting phytopathogen growth, particularly fungi, by limiting iron availability (Kumari et al., 2018). Furthermore, the production of siderophore enables plants to take iron even in the presence of other metals like nickel and cadmium (Beneduzi et al., 2012). In the current work, 5 bacterial isolates exhibited positive results for siderophore production, namely R4 (E. hormaechei), S7 (P. agglomerans), SA1 (S. gallinarum), MS3 (B. velezensis), and MS6 (K. sacchari). The different species from the genus Enterobacter and Pseudomonas are reported by Alexander and Zuberer (1991) to produce siderophores in varying concentrations. Bhattacharyya et al. (2020) reported comparable results for the production of siderophore in varying concentrations from 99.26% to 52.31 % by various microorganisms belonging to the genus Bacillus, Arthrobacter, Micrococcus, Pseudomonas, Staphylococcus, Ochrobactrum, and Exiguobacterium. Shariati et al. (2017) reported that TonB and fepA are genes that encode for siderophore receptors in the bacteria P. agglomerans

Hydrolytic enzymes, such as protease, amylase, chitinase, lipase, and cellulase, act as defenders to protect plants from diseases by degrading the cell wall of the plant’s pathogenic microorganisms (Zerihun et al., 2019). PGPB with the potential to produce one or more of these lytic enzymes can exhibit biocontrol against plant diseases caused by harmful phytopathogenic bacteria and fungi (Rasool et al., 2021). Sritongon et al. (2023) demonstrated a comparable result, that the PGPR Bacillus subtilis produced significant production of amylase and protease. The enzyme chitinases strengthen plant defense by targeting chitin, the primary structural component of fungal cell walls, thereby inactivating pathogenic fungi without harming the host plant. In addition to enhancing resistance, these enzymes also contribute to improved plant growth and yield (Kumar et al., 2018). The genus Serratia and Halomonas are reported to produce a range of hydrolytic enzymes, cellulases, amylases, lipases, proteases, ureases, and chitinases (Ouali et al., 2024).

The antagonistic activity of the PGPB is an important feature, as it can be used as a biocontrol agent. By producing hydrolytic enzymes, siderophores, antibiotics, and inducing systemic resistance in plants, the PGPB can inhibit the growth of phytopathogens. Several studies have demonstrated the potential of PGPB isolates from the genera Bacillus and Pseudomonas, particularly B. velezensis and P. fluorescens, as effective biocontrol agents (Arkhipov et al., 2023).

The application of PGPB enhances seed germination due to the release of phytohormones, which play a crucial role in cell division (Pérez-García et al., 2023). PGPB produces phytohormones such as IAA, gibberellins, and cytokinins, which stimulate cell division and elongation during germination. Similar results for seed germination enhancement by PGPB have been reported in numerous vegetables, including Daucus carota, Allium cepa, Capsicum annuum, and Solanum lycopersicum (Lobato Ureche et al., 2021; Miljaković et al., 2022; Monalisa and Roy, 2022). The biplot PCA demonstrated a positive correlation between the IAA production and PSI. The results are comparable to those previously reported by the authors Barbaccia et al. (2022) and Sahin et al. (2004), respectively. The results demonstrated that the seven PGPBs have exhibited PGP characteristics, such as nitrogen fixation, ammonia and IAA production, phosphate solubilization, HCN production, siderophore production, hydrolytic enzyme secretion, and antagonistic activity. Further field investigations of these PGPBs will reveal their impact on PGP, the mechanism of action, and demonstrate their potential contribution to sustainable agriculture.

This study offers valuable insights into plant growth-promoting rhizobacteria isolated from the millet rhizosphere, highlighting their significant role in enhancing tomato seed germination. The isolates exhibited multiple beneficial PGP traits, including nitrogen fixation, IAA production, ammonia production, phosphate solubilization, HCN production, siderophore production, hydrolytic enzyme production, and antagonistic activity, reflecting their potential in supporting plant growth and development. By harnessing the native microbial diversity of the millet rhizosphere, an underexplored but rich reservoir of agriculturally beneficial microbes, this present work highlights a sustainable approach to improve crop growth and development while reducing dependence on chemical fertilizers. Further investigation of the impact of these isolates on PGP will help to uncover the molecular mechanisms underlying their plant growth-promoting effects and assess their adaptability to diverse environmental conditions. Future work will prioritize optimization of bioformulation, consortia development, and field trials to develop targeted, crop-specific microbial inoculants capable of minimizing the usage of chemical fertilizers, enhancing soil fertility, and improving sustainable agriculture practices.