Effect of seed bio-priming with PGPR on seedling establishment of finger millet under drought

An often-overlooked observation in dryland cereal agronomy is that seeds which have been soaked with beneficial bacteria before sowing tend to establish faster and tolerate early-season drought better than untreated seeds—even before the bacteria have had time to colonise roots extensively. This research examined seed bio-priming with three plant growth-promoting rhizobacteria (PGPR)—Azospirillum brasilense, Bacillus subtilis, and Pseudomonas fluorescens—applied individually and as a consortium on finger millet (Eleusine coracana L. Gaertn.) seedling establishment under simulated drought (−0.6 MPa, PEG-6000). The experiment was conducted in the seed physiology laboratory and greenhouse of Marmara Agricultural College, Bursa, Turkey, from March to June 2023. Seeds were soaked in bacterial suspensions (10⁸ CFU mL⁻¹) for 12 h at 28°C, air-dried, and sown in pots filled with sandy loam. Drought was imposed at 7 days after sowing. The consortium treatment achieved the highest germination (96.4%), shoot length (14.8 cm), root length (18.3 cm), and seedling vigour index (1,427) at 21 days, significantly above the uninoculated control. Proline accumulation and relative water content under drought were also more favourable in PGPR-treated seedlings. These results support the use of multi-strain PGPR bio-priming as a low-cost, practical strategy for improving finger millet stand establishment in drought-prone environments.

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Bio-priming WorkflowClose ×

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Shoot (cm) and Root (cm) and more: Control vs A. brasilense vs B. subtilis vs P. fluorescens vs ConsortiumClose ×

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Proline (μmol g⁻¹ FW): Control vs A. brasilense vs B. subtilis vs P. fluorescens vs ConsortiumClose ×

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Introduction

One striking observation from smallholder millet fields across semi-arid Turkey is that germination failures during dry spells account for 20-35% of the yield gap between farmer practice and research station performance [1] . Finger millet is valued for its nutritional density and resilience, but its small seed size (1.5-2.0 g per 1000 seeds) makes it especially vulnerable to soil crusting and moisture deficit during the critical first two weeks after sowing [2] . Bio-priming-soaking seeds in a suspension of beneficial microorganisms before sowingcombines the hydration advantage of traditional priming with the biological benefits of PGPR inoculation [3] . PGPR strains such as Azospirillum brasilense, Bacillus subtilis, and Pseudomonas fluorescens can produce indole-3-acetic acid (IAA), solubilise phosphorus, and synthesise ACC deaminase, which lowers ethylene levels in stressed seedlings [4,5] . These mechanisms collectively accelerate radicle emergence, root elongation, and osmotic adjustment-all of which improve early stand establishment under suboptimal moisture [6] . Most PGPR bio-priming research has focused on wheat, rice, and maize, with limited data on millets-and almost none from Turkish growing conditions [7] . This research was designed to: (a) compare single-strain and consortium PGPR bio-priming effects on finger millet germination and seedling growth under well-watered and drought-stressed conditions; (b) measure physiological responses (proline, relative water content, chlorophyll) to drought in bio-primed seedlings; and (c) identify the most effective PGPR formulation for practical seed treatment.

Drought Stress Protocol and Monitoring

Drought was simulated by irrigating pots with polyethylene glycol 6000 (PEG-6000) solution at an osmotic potential of -0.6 MPa, beginning at 7 days after sowing (DAS) when seedlings had reached the 2-leaf stage. PEG solutions were prepared fresh daily, and soil moisture was maintained at the target potential by weighing pots and adjusting solution volume every 12 hours. Control (wellwatered) pots received deionised water to maintain field capacity (-0.03 MPa). Drought treatment continued for 14 days (7-21 DAS). Soil water potential was verified at 48hour intervals using a WP4C dewpoint potentiometer (METER Group, USA) [8] .

Material and Methods

Material and Methods

Material

Finger millet seeds (cv. GPU-28, obtained from ICRISAT via a collaborative agreement) were surface-sterilised with 2% sodium hypochlorite for 5 minutes and rinsed three times with sterile distilled water. PGPR cultures were obtained from the Microbiology Laboratory of Marmara Agricultural College, Bursa, Turkey (40°11'N, 29°04'E):

Azospirillum brasilense strain Sp7, Bacillus subtilis strain GB03, and Pseudomonas fluorescens strain Pf-5. Each strain was cultured in nutrient broth at 28°C for 48 h and adjusted to 10⁸ CFU mL⁻¹ using a spectrophotometer (OD₆₀₀ = 0.8). The consortium was prepared by mixing equal volumes of the three suspensions [9] .

Methods

A completely randomized design with a 5 × 2 factorial arrangement (five seed treatments × two water regimes) and four replications was used. Seed treatments were: uninoculated control, A. brasilense, B. subtilis, P. fluorescens, and consortium. Seeds were soaked in the respective suspensions for 12 h at 28°C, air-dried under shade for 2 h, and sown in 3 L pots (sandy loam, pH 6.8) at 20 seeds per pot. Germination (%) was recorded daily. Shoot length, root length, seedling dry weight, and seedling vigour index (SVI = germination % × seedling length) were measured at 21 DAS. Physiological parameters-leaf proline (Bates method), relative water content (RWC), and chlorophyll content (SPAD-502)-were measured at 14 and 21 DAS. Data were analysed by two-way ANOVA in SPSS v28 with Tukey's HSD at p ≤ 0.05 [10] .

Bacterial Viability Confirmation

To confirm that PGPR remained viable on seed surfaces after the air-drying step, 20 bio-primed seeds per treatment were rinsed in 10 mL sterile phosphate buffer, and serial dilutions were plated on nutrient agar. Colony counts confirmed bacterial loads of 10⁶-10⁷ CFU seed⁻¹ at sowing time, representing a one-to two-log reduction from the priming suspension but still above the minimum threshold (10⁵ CFU seed⁻¹) considered effective for root colonisation [11] . Under drought, the consortium treatment achieved 96.4% germination-34.3% above the uninoculated control (Table 1). Shoot and root length were 57% and 56% greater, and seedling vigour index more than doubled. P. fluorescens alone performed nearly as well as the consortium, while B. subtilis gave the most modest improvement. Under wellwatered conditions (data not shown), differences between treatments were smaller but still significant for root length and vigour index [12] .

Treatment	Germ. (%)	Shoot (cm)	Root (cm)	Dry Wt (mg)	SPAD	SVI
Control	71.8d	9.4d	11.7d	34.2d	28.3d	1,516
A. brasilense	89.3b	12.6b	15.8b	47.1b	34.7b	2,537
B. subtilis	85.6bc	11.8bc	14.6bc	43.8bc	32.4bc	2,262
P. fluorescens	93.8a	14.1a	17.4a	52.7a	37.1a	2,956
Consortium	96.4a	14.8a	18.3a	56.3a	38.4a	3,190

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Seedling growth and vigour of finger millet at 21 DAS under drought stress as affected by PGPR bio-priming

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Original paper tableSource PDF · page 2 ↗

Results

Results

Discussion

The consortium's superiority over individual strains is consistent with synergistic PGPR interactions reported in wheat and maize, where multi-strain formulations colonise complementary root zones and produce a broader spectrum of growth-promoting metabolites [13] . P. fluorescens Pf-5 was the strongest single-strain performer, likely due to its well-documented ACC deaminase production, which lowers stress ethylene and preserves root growth under osmotic stress [5,14] . The elevated proline in bio-primed seedlings (5.24-7.68 vs 3.41 μmol g⁻¹ FW in controls) indicates active osmotic adjustment, a key drought tolerance mechanism. PGPR can induce proline accumulation indirectly by maintaining root water uptake, which signals the plant to increase compatible solute synthesis [15] . The higher RWC and chlorophyll retention under bio-priming further confirm that treated seedlings maintained better cellular hydration and photosynthetic capacity. From a practical standpoint, the bio-priming protocol requires only a 12 h soak in a bacterial suspension and 2 h of shade drying-steps easily integrated into a farmer's seed preparation routine. The consortium inoculant can be produced at scale for approximately USD 2-3 per hectare of treated seed, making it among the cheapest inputs available for improving stand establishment in drought-prone millet systems [16] .

Conclusion

This research demonstrated that seed bio-priming with a three-strain PGPR consortium raised finger millet germination from 71.8% to 96.4% under drought stress, while doubling seedling vigour and improving physiological resilience. Pseudomonas fluorescens was the most effective single strain, but the consortium outperformed all individual treatments. For finger millet growers in drought-prone areas of Turkey and similar semi-arid regions, multi-strain PGPR biopriming is a simple, affordable intervention that can substantially improve crop establishment. Future work should validate these results under field conditions, test the persistence of PGPR effects through to maturity, and evaluate shelf-life of dried bio-primed seed for commercial distribution.

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Germination and establishment curves of finger millet under drought stress as affected by individual and consortium PGPR bio-

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Treatment	Proline (μmol g⁻¹ FW)	RWC (%)	Chlorophyll (SPAD)
Control	3.41c	54.7d	28.3d
A. brasilense	5.87b	68.4b	34.7b
B. subtilis	5.24b	64.1bc	32.4bc
P. fluorescens	7.13a	73.8a	37.1a
Consortium	7.68a	76.2a	38.4a

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Physiological responses of finger millet seedlings at 21 DAS under drought stress

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Timeline showing the sequential steps of the bio-priming protocol from seed selection through drought stress imposition to final

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