Genomic and Functional Characterization of Sunflower Endophytes Reveals Mechanisms of Drought Tolerance and Growth Promotion

Muritala Muhammed; Ayansina Segun Ayangbenro; Olubukola Oluranti Babalola

doi:10.1002/pld3.70184

. 2026 Jul 15;10(7):e70184. doi: 10.1002/pld3.70184

Genomic and Functional Characterization of Sunflower Endophytes Reveals Mechanisms of Drought Tolerance and Growth Promotion

Muritala Muhammed ¹, Ayansina Segun Ayangbenro ^1,², Olubukola Oluranti Babalola ^1,^3,^✉

PMCID: PMC13372015 PMID: 42459817

ABSTRACT

Drought severely limits sunflower ( Helianthus annuus L.) productivity, necessitating sustainable microbial strategies to enhance plant resilience. Endophytic bacteria modulate plant stress responses through diverse plant growth‐promoting (PGP) traits. This study characterized Pseudomonas protegens M4 and Citrobacter braakii M34 and evaluated their individual and combined effects on sunflower drought tolerance. Greenhouse experiments assessed inoculation with M4, M34, and their consortium (M4 + M34) under optimal (100% field capacity, FC) and drought conditions (75%–25% FC). Initial taxonomic identification based on 16S rRNA gene sequencing was further refined using whole‐genome sequencing (WGS) for high‐resolution classification and functional analysis. Genomic annotation and antiSMASH analysis revealed key PGP pathways in both strains, including enzymes involved in ethylene regulation, phosphate solubilization, biosynthesis of auxin‐like compounds, osmoprotectant production, and reactive oxygen species detoxification, alongside shared and strain‐specific secondary metabolite gene clusters. Under drought conditions, M4 primarily enhanced growth‐related traits such as shoot development and leaf area, whereas M34 was associated with physiological stress tolerance indicators. The consortium M4 + M34 exhibited synergistic effects, significantly increasing the chlorophyll content, relative water content, and proline accumulation while reducing electrolyte leakage. Principal component analysis further confirmed the consortium's superior performance. These findings highlight the potential of genomically and functionally characterized endophytic bacterial consortia as effective bioinoculant strategies for improving sunflower productivity under water‐limited conditions.

Keywords: drought stress, endophytic bacteria, microbial consortium, plant‐microbe interactions, whole‐genome sequencing

1. Introduction

Drought is a key environmental limitation of agricultural productivity worldwide and adversely affects crop performance and yield stability (Begna 2020; Khan et al. 2025). Climate predictions show that the frequency of drought will increase and its intensity will become more severe, diminishing arable land and freshwater supplies, thereby threatening global food security (Mahto and Mishra 2023). These challenges will reduce plant productivity and resilience, disrupting crop‐level food webs and agroecosystem functioning (Janni et al. 2024; Khan et al. 2025). Within cropping systems, drought stress inhibits the growth, photosynthesis, and reproduction of key cereals such as maize, wheat, and rice, resulting in substantial biomass reduction and yield losses (Hussain et al. 2019; Khan et al. 2025; Li et al. 2023; Santini et al. 2022).

Sunflower ( Helianthus annuus L.) is a globally significant oilseed crop; however, its productivity is highly sensitive to drought, particularly during critical growth stages such as flowering and seed filling. Drought stress has been reported to cause substantial reductions in biomass accumulation, oil content, and seed yield, thereby limiting its agronomic potential in water‐limited environments (Mahmood et al. 2021; Mostafa and Afify 2022).

Traditional approaches to reduce drought stress have emphasized conserving soil moisture, using irrigation technologies, and breeding for drought tolerance in crop varieties to improve water‐use efficiency. However, these approaches face economic, technological, and scalability limitations, particularly in resource‐limited regions (Fadiji et al. 2022a; Franco‐Navarro et al. 2025; Hassen et al. 2025; Wang et al. 2024).

Advances in microbiome research and high‐throughput sequencing technologies have opened avenues for cost‐effective strategies to modulate plant‐associated microbial communities, offering a promising approach to ameliorate the influence of drought stress on crop productivity (Agunbiade and Babalola 2024; Sharma et al. 2025). Plant growth‐promoting (PGP) endophytic Proteobacteria, particularly Pseudomonas species, enhance plant performance under water‐limited conditions by improving root architecture, nutrient uptake, and antioxidant defense systems (Abideen et al. 2022). Emerging evidence also highlights the functional significance of Citrobacter species in abiotic stress alleviation through osmolyte production and multiple PGP traits (Adedayo et al. 2022; Chen et al. 2025; Liu et al. 2021). Pseudomonas genes were demonstrated to improve drought mitigation and yield in wheat and maize through siderophore‐mediated iron acquisition, IAA‐induced root development, and antimicrobial metabolite production (Adedeji and Babalola 2020; Giannelli et al. 2024; Olanrewaju and Babalola 2019). Similarly, Citrobacter species increase resilience in tomato and cactus hosts through phosphate solubilization, IAA production, and osmoprotectant synthesis, thereby improving nutrient acquisition and reducing ethylene‐mediated stress (Eke et al. 2019; Liu et al. 2021). Collectively, these traits contribute to improved biomass accumulation and yield stability in drought‐stressed crops (Borker et al. 2024; Chukwuneme et al. 2020).

In this study, we investigated the genomic and functional traits of endophytic bacteria isolated from sunflower and assessed their capacities to enhance plant performance under drought stress. By integrating genome‐based analysis with physiological and biochemical assessments, this work aims to provide insights into plant interactions under water‐limited conditions. We hypothesize that functionally complementary endophytic bacterial consortia can enhance sunflower drought tolerance more effectively than individual strains through coordinated growth‐promoting and stress‐responsive mechanisms.

2. Experimental Procedures

2.1. Isolation of Sunflower Endophytic Proteobacteria

A total of 36 healthy sunflower plants (cultivar PAN 7080) at the vegetative stage were randomly collected from farmland in Delareyville, North West Province, South Africa (27.11828° S, 25.88914° E). To ensure a representative sampling of field variability, plants were collected along three diagonal transects across the field with 12 plants per transect at approximately equal intervals. The root samples were excised, placed in sterile polyethylene bags, and transported to the laboratory under ice (4°C) for further processing. In the laboratory, root samples were thoroughly washed under running water to remove adhering soil and debris and then preserved under aseptic conditions prior to surface sterilization to eliminate epiphytic microorganisms. Surface sterilization was performed sequentially by immersion in 70% ethanol solution for 3 min under continuous agitation to ensure uniform decontamination. This step was followed by soaking in 2% NaOCl for 5 min. A final 30‐s rinse in 70% (v/v) ethanol was performed, and sterile deionized water was used to rinse the samples to remove residual chemicals. The effectiveness of surface sterilization was assessed by plating 0.1 mL of the final rinse water onto Luria–Bertani (LB) agar, incubating at 28°C for 48 h, and examining for bacterial growth. The absence of microbial growth confirmed effective surface sterilization. Sterilized root samples were aseptically sectioned into approximately 1‐cm segments, pooled, and homogenized using a sterile mortar and pestle to generate a composite sample for endophytic bacterial isolation, thereby enhancing recovery of culturable endophytic diversity. One gram of homogenized tissue was suspended in 1 mL of sterile 1‐M phosphate‐buffered saline (PBS, pH 7.0) and ground until a uniform suspension was obtained. Serial tenfold dilutions were prepared up to 10⁻⁶. From the 10⁻³ dilution, 0.1‐mL aliquots were pour‐plated onto King's B (KB) agar and Pseudomonas Isolation Agar, according to the procedures outlined by Khamwan et al. (2018). King's B medium was used to support the growth and fluorescent pigment production of Pseudomonas species, whereas PIA was employed as a selective medium to suppress nontarget bacteria and enrich Pseudomonas populations.

Following inoculation, the plates were incubated at 28°C for 24 h, and individual, well‐separated colonies were selected and purified through repeated subculturing. Pure isolates were maintained on agar slants at 4°C for further characterization.

2.2. Morphological, Culture, and Biochemical Profiling of Endophytic Proteobacteria

Following the cultivation of several subcultures on LB agar, the cultural and biochemical attributes of both bacterial isolates were assessed. Microscopic examination of each isolate was performed via a light microscope (ECLIPSE E200, Nikon Japan) to determine the cellular morphology. A series of biochemical assays, including oxidase, catalase, and indole acetic acid production, was conducted in accordance with established protocols detailed by Bashir et al. (2020).

2.3. Assessment of Drought Tolerance in Endophytic Proteobacteria Using Polyethylene Glycol (PEG8000)

The ability of strain M4 and M34 to tolerate osmotic stress was evaluated via the use of polyethylene glycol (PEG 8000) as the osmotic agent. Bacterial proliferation was assessed in nutrient broth supplemented with varying concentrations of PEG8000 (0%–30% (w/v)) to simulate gradually decreasing water availability. Culturing was conducted for 24 h at 28°C under continuous agitation at 180 rpm. Cell growth was evaluated spectrophotometrically at 600 nm (Thermo Spectronic; Merck, South Africa), and growth patterns under osmotic stress were examined as described by Sandhya et al. (2009).

2.4. Evaluation of the PGP Properties of Endophytic Bacteria

PGP traits of endophytic bacteria were evaluated using established assays for ammonia production, phosphate solubilization, siderophore synthesis, hydrogen cyanide generation, indole‐3‐acetic acid (IAA) production, ACC deaminase activity, and exopolysaccharide production. Ammonia production was evaluated by culturing bacteria in peptone water followed by colorimetric detection using Nessler's reagent as described by Cappuccino and Sherman (1992). Phosphate solubilization was assessed on Pikovskaya's agar containing insoluble phosphate sources following Pikovskaya (1948), siderophore production was determined using the chrome azurol S (CAS) assay developed by Schwyn and Neilands (1987) with FeCl₃.6H2O as the iron source. IAA production was evaluated colorimetrically in tryptophan‐supplemented medium using Salkowski reagent according to Gordon and Weber (1951). Hydrogen cyanide production was detected qualitatively using alkaline picrate‐impregnated filter paper as described by Bakker and Schippers (1987). ACC deaminase activity was assessed qualitatively based on the ability of bacterial isolates to grow on minimal salt medium supplemented with 1‐aminocyclopropane‐1‐carboxylate (ACC) as the sole nitrogen source, following the method of Penrose and Glick (2003). Exopolysaccharide production (EPS) was evaluated based on the formation of mucoid colonies on sucrose‐enriched medium following standard screening protocols for EPS‐producing bacteria (Costa et al. 2018). All assays were conducted in triplicate to maintain experimental consistency. Detailed experimental procedures are provided in the Supporting Information.

Based on confirmed PGP traits and tolerance to osmotic stress under laboratory conditions, strains M4 and M34 were selected as candidate inoculants for subsequent greenhouse experiments.

2.5. Molecular Identification of Endophytic Proteobacteria

Genomic DNA was extracted from axenic bacterial cultures using the Zymo Fungal/Bacterial DNA Miniprep Kit (Zymo Research, USA) according to the manufacturer's instructions. DNA quality and concentration were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).

Initial taxonomic identification of the isolates was performed using 16S rRNA gene sequencing (Sanger sequencing). The resulting sequences were deposited in the NCBI GenBank database under accession numbers PQ428896 (M4) and PQ428888 (M34). To achieve higher resolution taxonomic classification and enable comprehensive functional characterization, whole‐genome sequencing (WGS) was subsequently performed. Genome sequencing of the isolates was performed at Novogene Co. Ltd. (Singapore) using the Illumina NovaSeq X Plus sequencing platform. Library preparation, including enzymatic fragmentation, adapter ligation, size selection, and PCR enrichment, was performed using the Nextera DNA Flex platform (Illumina, USA). Library purification was carried out via AMPure XP magnetic beads (Beckman Coulter, USA), followed by fragment size analysis via an Agilent Fragment Analyzer (Agilent Technologies, USA) and quantification via a Qubit fluorometer (Thermo Fisher Scientific, USA). Sequencing produced bidirectional reads with a read length of 150 bp from both ends of each DNA fragment.

The raw reads were processed via the KBase platform (Arkin et al. 2018). Quality evaluation was carried out via FastQC (v0.12.1) (Andrews 2010), and trimming of adapters and low‐quality bases was conducted via Trimmomatic (v0.39) (Bolger et al. 2014). Genome assembly was conducted with the SPAdes assembler (v3.15.3) following Prjibelski et al. (2020), and genome quality, comprising completeness and possible impurities, was assessed with CheckM (v1.0.18) as described by Parks et al. (2015). Biosynthetic gene clusters (BGCs) were detected via antiSMASH (v8.0.2) (Blin et al. 2025), with gene prediction and functional annotation of the assembled genomes performed via the Prokaryotic Genome Annotation Pipeline (PGAP), as outlined by (Tatusova et al. 2016). Circular representations of the assembled genomes were visualized with CGView (v3.55.17) (Stothard and Wishart 2005). Unless stated otherwise, default parameters were applied.

Phylogenomic analysis was conducted using the BV‐BRC platform based on conserved single‐copy core genes from assembled genomes and closely related reference strains. Maximum‐likelihood trees were inferred within the BV‐BRC, with bootstrap support values (%) indicated at the nodes and branch lengths representing substitutions per site (Olson et al. 2023).

2.6. Greenhouse Evaluation of Bacterial Inoculation Effects on Sunflower Growth Under Contrasting Water Regimes

2.6.1. Soil Collection and Processing for Greenhouse Trials

The soil used in the greenhouse study was obtained from an agricultural site situated adjacent to the Animal Health Department at North‐West University, Mahikeng Campus (25°49′26.256″ S, 25°36′31.536″ E). The surface soil samples were collected from the 0–20‐cm layer. Coarse debris, including plant residues and stones, was manually removed. The soil was air‐dried and then oven‐dried at 70°C for 48 h under ambient laboratory conditions. Finally, the dried soil was sieved through a 2‐mm mesh to obtain a uniform particle size. The prepared soil was sterilized by autoclaving at 121°C for 15 min in an SA‐300VL autoclave (Taiwan). Following a 2‐day cooling period, 9 kg of the prepared soil was transferred into each of 128 sterile plastic containers (30 × 29 cm) for the greenhouse experiment.

2.6.2. Measurement of Experimental Soil Moisture Retention

Soil water‐holding capacity was determined gravimetrically using a saturation–drainage approach adapted from Nelson et al. (2024). Briefly, air‐dried and sieved soil, approximately 100 g, was saturated with deionized water, allowed to drain freely under gravity for 24 h, and weighed before and after oven drying at 105°C to constant mass. Gravimetric water‐holding capacity was calculated from the difference between wet and dry weights. Detailed procedures and calculations are provided in Supporting Information.

2.6.3. Seed Sterilization and Bacterial Inoculum Preparation

Sunflower seeds of two genotypes were used: PAN7080 (drought tolerant; OBARO, Potchefstroom, South Africa) and SSR‐Faithful to Nature (drought‐susceptible; Faithful to Nature, online supplier, South Africa). Seeds were surface‐sterilized via a sequential treatment: immersion in 70% (v/v) ethanol for 5 min, followed by immersion in 2% (v/v) NaOCl for 2 min, and a final rinse in 70% (v/v) ethanol for 1 min. The residual disinfectants were removed by washing the seeds three times with sterile distilled water.

To prepare the bacterial inoculum, strains M4 and M34 were cultured separately in 500 mL of TSB at 25°C with constant shaking at 120 rpm for 3 days. After incubation, the cells were pelleted by centrifugation at 10,000 rpm for 10 min, rinsed twice with autoclaved deionized water, and then reconstituted in 0.01‐M PBS at pH 7. The bacterial suspensions were standardized to an optical density at 600 nm (OD₆₀₀) of 1.5, as measured with a spectrophotometer (Thermo Spectronic, Merck, SA), following Farhat et al. (2023) and Zhang et al. (2023).

Pregerminated sunflower seeds were submerged in the respective bacterial inocula and left to soak for 12 h to enable successful colonization. The plants were subsequently allowed to air‐dry overnight under sterile conditions and planted. Uninoculated controls received identical treatments, except that sterile distilled water was used in place of the bacterial inoculants.

2.6.4. Experimental Design and Drought Stress Application

Five sunflower ( H. annuus ) seeds were sown per pot containing 9 kg of sterilized soil, and seedlings were thinned to one healthy plant per pot 2 weeks after emergence. The experiment followed a completely randomized design (CRD) in a 2 × 4 × 4 factorial arrangement with eight replicates per treatment. The factors included: two sunflower genotypes (PAN7080), drought tolerant; and SSR‐Faithful to Nature, drought‐susceptible (SSR); (ii) four water regimes (100% FC [860 mL day⁻¹], 75% FC [645 mL day⁻¹], 50% FC [430 mL day⁻¹], and 25% FC [215 mL day⁻¹]); and (iii) four bacterial treatments (uninoculated control, M4, M34, and a combined M4 + M34 consortium).

Drought stress was imposed 2 weeks after germination and maintained gravimetrically by daily water replenishment to achieve target field capacity levels. Bacterial inoculation was performed in the second and fourth weeks after drought initiation by applying 5 mL of freshly prepared bacterial suspension to the root zone of each plant using sterile pipettes. For the consortium treatment, equal volumes of each bacterial suspension were combined to obtain a total inoculum volume of 5 mL per plant while maintaining equivalent cell density to the single‐strain treatments (Farhat et al. 2023; Zhang et al. 2023). Plants were grown under controlled screenhouse conditions at 25°C (night) and 30°C (daylight) with a 14‐h light/10 h dark photoperiod.

2.6.5. Determination of Physiological Parameters

Physiological parameters were evaluated using standard methods. Relative water content (RWC) was assessed gravimetrically using fresh (FW), turgid (TW), and dry (DW) leaf weights following the method of Barrs and Weatherley (1962). Chlorophyll content in sunflower leaves was quantified using the dimethyl sulfoxide (DMSO) extraction procedure of Hiscox and Israelstam (1979), with absorbance measured at 645 and 663 nm, and chlorophyll a, chlorophyll b, and total chlorophyll were calculated according to Arnon (1949). Proline content was determined following Bates et al. (1973). Absorbance was measured at 520 nm, and proline content was calculated from a standard curve and expressed as μmol g⁻¹ FW. Electrolyte leakage was measured as an indicator of membrane integrity by comparing conductivity before and after tissue disruption, following Hatsugai and Katagiri (2018). Detailed procedures are provided in Supporting Information.

2.6.6. Data Collection and Bacterial Reisolation

The plants were harvested at physiological maturity, which was defined as 120 days after sowing. Prior to harvest, plant growth parameters were monitored every 2 weeks throughout the experiment. The morphological characteristics of the sunflower plants, including shoot length (cm), total leaf count, leaf length and width (cm), and leaf surface area (cm²), were measured throughout the experiment. Simultaneously, physiological parameters, including the chlorophyll concentration index, RWC, and proline accumulation, were assessed using methods previously described. Upon completion of the experimental period, the plants were gently removed from the soil and the roots were thoroughly rinsed under running water to remove attached soil particles. The fresh masses of the aerial and root tissues were determined via a calibrated analytic balance. Afterward, the plant tissue was dried in an oven at 70°C until a constant weight was reached, and the dry weight (g) was measured.

To verify endophytic colonization, sunflower roots were surface sterilized to eliminate epiphytic microorganisms prior to bacterial reisolation. The sterilized root tissues were aseptically macerated and serially diluted, and aliquots (10⁻³ dilution) were plated on PIA and King's B agar media 4 weeks after inoculation. Recovered isolates were compared with the original inoculated strains based on colony morphology, biochemical characteristics, and PCR amplification using strain‐specific primers, which consistently confirmed their similarity. To further support colonization, a complementary hydroponic assay was conducted under controlled conditions to minimize external microbial interference. The consistent recovery of strains with comparable phenotypical and molecular characteristics across both soil‐based and hydroponic systems confirmed successful endophytic colonization.

2.7. Data Analysis

Analysis of variance (ANOVA) was performed within the general linear model framework. Post hoc comparisons of treatment means were conducted using Duncan's multiple range test (DMRT) at the 95% confidence level, implemented in SAS software version 9.1. To identify and quantify the contributions of key parameters to the variability observed across treatments, a principal component analysis (PCA) was carried out using Canoco software, version 5.1. Tables and figures for this study are provided in Supporting Information.

3. Results

3.1. Morphological and Biochemical Characterization of Isolates

The cultural and biochemical characteristics of Pseudomonas protegens M4 and C. braakii M34, isolated from the sunflower endosphere, are summarized in Table S1. The two endophytic strains are Gram‐negative, rod‐shaped bacteria with creamy colony morphology and exhibit positive catalase, oxidase, and citrate‐utilizing reactions. Both isolates utilized galactose, fructose, and maltose as carbon sources but did not metabolize mannitol. Optimal growth conditions differed slightly between the isolates, with P. protegens M4 showing optimal growth at 25°C–30°C and pH 6–8, whereas C. braakii M34 exhibited optimal growth at 35°C–40°C and pH 7–8. These preliminary phenotypic characteristics provided a basis for further functional and genomic characterization of the isolates.

3.2. In Vitro PGP Properties and Osmotic Stress Tolerance of M4 and M34

The PGP traits of the isolates are presented in Table 1. Both strains exhibited multiple beneficial traits, although their magnitudes differed. P. protegens M4 exhibited higher phosphate‐solubilizing ability and indole‐3‐acetic acid (IAA) production compared with C. braakii M34. Both isolates produced exopolysaccharide, ammonia, 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase, and hydrogen cyanide, whereas no detectable siderophore production was observed under the experimental conditions. The strains also demonstrated tolerance to osmotic stress, maintaining growth across increasing polyethylene glycol (PEG)‐induced water potentials (Table 2). These traits indicate their capacity to function under water‐limited conditions and contribute to plant stress mitigation. These results show the functional potential of the isolates and support their further evaluation under greenhouse drought conditions.

TABLE 1.

Qualitative assessment of P. protegens M4 and Citrobacter braakii M34 for plant growth‐promoting traits.

Trait	M4	M34
Phosphate solubilization	++	+
IAA production	++	+
Exopolysaccharide production	+	+
Ammonia production	+	+
Siderophore production	−	−
ACC deaminase	+	+
Hydrogen cyanide	+	+

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Note: + = positive; ++ = strongly positive; − = negative.

TABLE 2.

Effect of PEG 8000 on P. protegens M4 and Citrobacter braakii M34 growth (OD600).

PEG (%)	M4 (OD600 ± SD)	M34 (OD600 ± SD)
0	1.07 ± 0.04^a	0.86 ± 0.01a
5	0.91 ± 0.01^b	0.66 ± 0.01^b
10	0.86 ± 0.02^b	0.54 ± 0.06^c
15	0.64 ± 0.02^c	0.47 ± 0.02^c
20	0.37 ± 0.00^d	0.22 ± 0.01^d
25	0.16 ± 0.01^e	0.15 ± 0.02^e
30	0.08 ± 0.00^f	0.06 ± 0.00^f

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Values with different superscript letters differ significantly at p ≤ 0.05 according to DMRT.

3.3. Genomic Features and Functional Annotation of Bacterial Isolates M4 and M34

WGS confirmed the taxonomic identity of strains M4 and M34 as Pseudomonas protegens and C. braakii , respectively. High‐quality genome assemblies were obtained, with genome sizes of 6,900,000 and 4,900,000 bp, respectively (Table 3; Figures S1–S5). Both genomes contained genes associated with plant growth promotion, nutrient acquisition, and stress adaptation. Functional annotation revealed pathways related to phytohormone production, phosphate solubilization, siderophore production, and osmotic stress tolerance, supporting their potential roles in improving plant performance under drought conditions (Tables S2 and S3). These genomic insights provide a molecular basis for the observed plant growth‐promotion and stress tolerance traits.

TABLE 3.

Genome properties of P. protegens M4 and Citrobacter braakii M34.

Genomic parameters	M4	M34
Raw reads (bp)	9,080,964	8,395,238
Reads after trimming (bp)	8,813,994	8,213,592
Genomic size (Mb)	6.9	4.9
Number of contigs	13	12
GC contents (%)	63.50	52.00
N ₅₀ value (kb)	810.2	2700
L ₅₀ value (kb)	3	1
tRNA	51	77
rRNA	1	3
Total predicted genes	6237	4740
Protein‐coding genes	6112	4571
Pseudo genes	68	78
CDSs	6180	4649

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3.3.1. Predicted Secondary Metabolite Gene Clusters

AntiSMASH analysis revealed both shared and strain‐specific secondary metabolite BGCs in the two isolates, including arylpolyene, terpene–precursor, and NRP‐metallophore clusters. In addition to these conserved BGCs, P. protegens M4 harbored several additional clusters, such as protegencin‐like polyyne, β‐lactone, pyoluteorin‐like type I PKS, 2,4‐diacetylphloroglucinol (DAPG), RiPP‐like type III PKS, and orfamide A–like nonribosomal peptide clusters. The complete profiles of predicted BGCs identified in both genomes are provided in Tables S4 and S5. The absence of detectable siderophore production under CAS assay conditions does not preclude iron acquisition potential, as the expression of metallophore‐related gene clusters is often condition‐dependent and influenced by environmental or host‐associated cues.

3.4. Effects of Bacterial Inoculation on Sunflower Growth and Physiology Under Different Water Regimes

Bacterial inoculation significantly improved sunflower growth, physiological performance, and yield‐related traits across all water regimes, as indicated by the statistical analyses in Tables S6–S9. A qualitative synthesis of the overall response patterns across treatments and water regimes is summarized in Table 4.

TABLE 4.

Qualitative synthesis of sunflower growth and physiological responses to bacterial inoculation under drought stress.

Water regime	Treatment	Plant height	Leaf area	Proline accumulation	Electrolyte leakage	Relative water content
100%‐FC	Control	Reference	Reference	Low	Moderate	High
	M4	↑	↑↑	Low	↓	↑
	M34	↑	↑	Low	↓	↑
	M4 + M34	↑	↑	↑↑	↓↓	↑↑
75%‐FC	Control	↓	↓	Moderate	↑	↓
	M4	↑↑	↑↑	Moderate	↓	↑
	M34	↑	↑	Moderate	↓	↑
	M4 + M34	↑	↑	↑↑	↓↓	↑↑
50%‐FC	Control	↓↓	↓↓	Moderate	↑↑	↓↓
	M4	↑	↑	Moderate	↓	↑
	M34	↑	↑	Moderate	↓	↑
	M4 + M34	↑	↑	↑↑	↓↓	↑↑
25%‐FC	Control	↓↓↓	↓↓↓	Low	↑↑↑	↓↓↓
	M4	↑	↑	Moderate	↓	↑
	M34	↑	↑	Moderate	↓	↑
	M4 + M34	↑↑	↑↑	↑↑↑	↓↓↓	↑↑

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Under well‐watered conditions (100% FC), inoculated plants exhibited greater biomass accumulation, leaf area, and chlorophyll content than the uninoculated control (Table S6).

Under moderate drought (75% FC) and severe (50% FC) drought stress, inoculated treatments maintained higher growth and physiological stability, as reflected by increased RWC and chlorophyll levels. Proline accumulation was enhanced in inoculated plants, indicating enhanced osmotic adjustment, whereas electrolyte leakage was reduced, suggesting improved membrane integrity (Tables S7 and S8).

Under extreme drought (25% FC), all plants showed reduced growth; however, inoculated treatments, particularly the consortium, retained significantly higher physiological performance compared to the control. The consortium consistently outperformed single‐strain inoculations across traits, indicating a synergistic effect on plant growth and stress tolerance. Genotype‐specific responses were also observed, with the drought‐tolerant cultivar maintaining higher baseline performance across treatments (Table S10), while still benefiting from bacterial inoculation. To further explore the relationships among measured traits and treatments, multivariate analysis was performed.

Table 4 provides a qualitative synthesis of the overall trends in sunflower growth and physiological and biochemical responses following inoculation with Pseudomonas protegens M4, C. braakii M34, and their consortia under drought stress. The summarized trends are derived from the datasets analyzed statistically in Tables S7–S10. The arrows indicate directional responses relative to the uninoculated control (↑, increase; ↓, decrease; ↑↑/↓↓ stronger response). No independent statistical analyses were performed directly for this table.

3.5. Multivariate Analysis of Treatment Effects Under Drought Stress

PCA across different water regimes revealed progressive differentiation of treatment effects with increasing drought severity (Figure 1; Figures S6–S8). Under well‐watered conditions (100% field capacity), treatments showed relatively overlapping clustering, indicating limited divergence in stress‐induced variation (Figure S6). However, under mild, moderate, and severe drought, distinct clustering emerged along the first principal component, reflecting treatment‐driven differences in physiological and growth responses (Figure S7 and Figure S8). Under extreme drought (25% field capacity), the first two principal components explained 88.82% and 6.68% of the total variance, respectively, for a cumulative 95.50% (Figure 1). PC1 explained 88.82% of the total variance, indicating strong separation among treatments.

Principal component analysis (PCA) of sunflower growth and physiological traits under severe drought stress (25% field capacity). PC1 and PC2 explained 88.82% and 6.88% of the total variance, respectively (95.50% cumulative), indicating that variation was largely driven by a single dominant axis associated with treatment and drought stress responses under severe water limitation.

The PCA biplot showed that inoculated and noninoculated plants were clearly separated, with the consortium treatment (MC) forming a distinct cluster. Individual inoculations showed an intermediate position between the control and consortium treatments. Trait loadings indicated that Pseudomonas protegens M4 was primarily associated with growth‐related parameters, whereas Citrobacter braakii M34 aligned with physiological stress‐related traits contributing to variation across both principal components. Consortium treatment occupied an intermediate position, suggesting functional complementarity between growth promotion and drought tolerance mechanisms. Traits, including RWC, proline accumulation, chlorophyll content, and electrolyte leakage, contributed strongly to the observed variation and were major determinants of treatment differentiation. A gradient distribution of samples along the principal components corresponded to decreasing field capacity (100%–25%), highlighting water availability as a primary driver of physiological responses. Traits abbreviations are provided in Figure S6.

4. Discussion

Agricultural systems are increasingly exposed to environmental constraints, particularly drought, which disrupts plant physiological processes, limits biomass accumulation, and reduces yield stability under changing climate conditions (Begna 2023; Bhagat et al. 2021; Khan et al. 2025). These challenges highlight the need for sustainable strategies that enhance crop resilience by reducing reliance on chemical inputs. In this regard, plant growth‐promoting bacteria (PGPB) represent a promising approach due to their capacity to modulate plant responses to stress through multiple coordinated mechanisms rather than single functional traits (Ferioun et al. 2025; Kumawat et al. 2023).

This study demonstrates that endophytic bacterial inoculation enhances sunflower performance under drought stress through coordinated physiological and functional mechanisms. Integration of genomic, in vitro, and greenhouse data revealed that both Pseudomonas protegens M4 and C. braakii M34 possess multiple traits contributing to plant growth promotion, nutrient acquisition, and stress tolerance under both optimal and drought conditions (Tables 1, 2, 3; Tables S2–S5).

Genomic analysis confirmed the presence of genes associated with phytohormone production, nutrient acquisition, and stress mitigation, supporting the functional capabilities observed in vitro (Table 3). PGP traits such as indole‐3‐acetic acid (IAA) biosynthesis, ACC deaminase activity, phosphate solubilization, and siderophore production enhance nutrient uptake and root development, thereby improving plant growth under stress conditions. Under drought stress, these traits are complemented by mechanisms that regulate osmotic balance and protect cellular integrity, which are critical for maintaining physiological stability. The agreement between genomic potential and phenotypic expression strengthens the functional relevance of these isolates as bioinoculant candidates, consistent with previous reports demonstrating that genomic traits in endophytic bacteria are closely linked to their plant‐beneficial functions and stress adaptation capacity (Babalola et al. 2026; Lee et al. 2024; Rigerte et al. 2025; Semenzato and Fani 2024).

Both strains demonstrated tolerance to osmotic stress, although M4 exhibited greater resilience under higher PEG concentrations, suggesting differences in intrinsic stress adaptation capacity (Table 2). This may be associated with its larger genome and the presence of diverse BGCs (Table S2), including those related to antimicrobial activity, rhizosphere competence, and metabolite production (de Lima et al. 2024; Fadiji et al. 2022b). In contrast, M34 possessed genomic features more closely associated with nutrient acquisition and stress mitigation. These distinctions indicate that the two strains occupy complementary functional niches, which is critical for understanding their combined effects. The detection of genes involved in osmolyte biosynthesis and antioxidant defense in both genomes further supports their potential to persist and function under water‐limited conditions (Tables S2 and S3). Notably, neither genome contained a canonical acdS gene; however, both strains exhibited ACC deaminase activity in vitro. This suggests the presence of alternative enzymatic pathways or highly divergent acdS, possibly reflecting highly divergent acdS homologs, as previously reported in plant‐associated bacteria (Shahid et al. 2023). The absence of detectable siderophore production in vitro does not preclude iron acquisition, as expression is often context‐dependent and influenced by environmental or host‐derived cues (Mai et al. 2022; Song et al. 2024; Srivastava et al. 2022).

The larger genome of P. protegens M4 suggests greater functional versatility for host colonization, environmental adaptation, and metabolite production (Table 3). Comparative genome mining revealed conserved BGCs, such as arylpolyene, terpene–precursor, and NRP‐metallophore clusters, which are common in endophytes and are associated with persistence under stress (Mukherjee et al. 2023; Raj et al. 2023; White and Torres 2010). M4 harbored additional BGCs encoding protegencin‐like polyyne, β‐lactone, pyoluteorin‐like type I PKS, 2,4‐diacetylphloroglucinol (DAPG), RiPP‐like type III PKS, and orfamide A–like NRPS, contributing to antagonistic activity, root colonization efficiency, and microbial competitiveness (Balthazar et al. 2022; Cesa‐Luna et al. 2023; Takeuchi and Someya 2019). In contrast, M34 BGCs were dominated by enterobactin‐type NRP‐metallophores and azole‐containing RiPP clusters, which is consistent with a functional strategy centered on nutrient acquisition and stress mitigation. The predicted BGCs for M4 and M34 are summarized in Tables S4 and S5.

Greenhouse experiments demonstrated that inoculation with strains M4 and M34 significantly enhanced sunflower growth, physiological stability, and yield‐associated traits under both well‐watered and drought conditions (25%–75% FC). Inoculated plants exhibited increased biomass accumulation, higher chlorophyll content, improved RWC, enhanced proline accumulation, and reduced electrolyte leakage, indicating improved osmotic adjustment, membrane stability, and protection against oxidative damage, which are key determinants of drought tolerance (Anjum et al. 2017; Wang et al. 2019).

Importantly, the consortium treatment consistently outperformed single‐strain inoculations, indicating a synergistic interaction between the two isolates. This enhanced performance can be attributed to functional complementarity between the two strains (Babalola et al. 2026). Pseudomonas protegens M4 was primarily associated with growth‐related traits such as phytohormone production and phosphate solubilization, which enhance nutrient acquisition and biomass accumulation. In contrast, C. braakii M34 was more strongly associated with physiological stress‐responsive traits, including osmotic adjustment and maintenance of cellular integrity under drought conditions, as reflected in reduced electrolyte leakage and increased proline accumulation.

When coinoculated, these strains likely operated through coordinated mechanisms, enabling simultaneous optimization of growth and stress tolerance processes that are often physiologically constrained under drought conditions. Furthermore, this complementary interaction improves rhizosphere competence and root colonization efficiency, thereby amplifying plant‐microbe interactions. Such synergistic interactions have been widely reported in multistrain PGPR systems, where combined metabolic capabilities enhanced plant performance compared to single inoculants (Anjum et al. 2017; Wang et al. 2019).

Multivariate analysis further supported these findings, with principal component analysis revealing distinct trait association patterns among treatments. Trait loadings indicated that Pseudomonas protegens M4 was primarily associated with growth‐related parameters, whereas C. braakii M34 aligned with stress‐responsive traits, highlighting functional differences in sunflower responses to microbial inoculation under drought stress. The consortium treatment clustered distinctly under severe drought conditions, indicating a coordinated enhancement of plant physiological responses.

Genotype‐specific responses were evident, with the drought‐tolerant cultivar maintaining higher baseline performance across treatments. However, both genotypes showed significant improvement following bacterial inoculation, indicating that microbial intervention enhances drought tolerance beyond inherent genetic capacity (Table S10). Collectively, these findings show that integrating genomic and functional characterization with multistrain inoculation strategies provides a robust framework for developing effective microbial solutions for drought‐stressed agroecosystems.

5. Conclusions

Endophytic bacterial isolates enhanced sunflower performance under drought stress, with the consortium treatment producing the most consistent improvements. Combined physiological, biochemical, and genomic evidence suggests that these effects are mediated by coordinated mechanisms, including improved water status, enhanced nutrient acquisition, and regulation of the stress response. The enhanced performance observed under coinoculation supports the role of functional complementarity among strains in promoting plant resilience under drought stress. These findings indicate that endophytic microbial consortia have potential as sustainable tools for improving crop performance under drought. Despite these promising findings, some limitations are acknowledged. Although endophytic colonization was supported by reisolation and PCR‐based confirmation, the absence of whole‐genome resequencing limits strain‐level resolution and definitive verification of colonization. Additionally, although greenhouse conditions provide controlled insights into plant‐microbe interactions, field validation is required to confirm the consistency and scalability of these effects under variable environmental conditions.

Author Contributions

Muritala Muhammed: methodology, investigation, data curation, formal analysis, validation, writing – original draft, writing – review and editing. Ayansina Segun Ayangbenro: formal analysis, validation, investigation, visualization, data curation, writing – review and editing. Olubukola Oluranti Babalola: conceptualization, formal analysis, investigation, validation, supervision, project administration, resources, funding acquisition, validation, visualization, funding, writing – review and editing.

Funding

O.O.B. recognizes the National Research Foundation (NRF) South Africa for grants (UID123634 and UID132595) that support this research.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Phylogenomic placement of Citrobacter braakii M34 (A) and Pseudomonas protegens M4 (B) based on whole‐genome sequences. Phylogenomic trees were reconstructed using the maximum‐likelihood method implemented in the BV‐BRC platform. Bootstrap support values (%) are indicated at the nodes. The proportional length of each branch represents evolutionary divergence quantified as substitutions per nucleotide site. The strains characterized in the present study are marked in red. Reference strains, including type strains (indicated by “T”), were included for accurate taxonomic placement. Scale bars indicate nucleotide substitutions per site.

Figure S2: Circular genome visualization of whole genome of P. protegens M4.

Figure S3: Subsystem category distribution of key protein‐coding genes of P. protegens in M4.

Figure S4: Circular genome visualization of whole genome of C. braakii M34.

Figure S5: Subsystem category distribution of key protein‐coding genes of C. braakii in M34.

Figure S6: Principal component analysis (PCA) biplot of sunflower growth, agronomic, and physiological traits under well‐watered conditions (100% field capacity) with bacterial inoculations. The first two principal components (PC1 and PC2) explained 55.21% and 36.84% of the total variance, respectively (92.05% cumulative), indicating strong representation of traits variability. Treatments (CTRL, M4, M34, and MC) are distributed based on trait associations, highlighting differences in plant performance under nonstress conditions. Trait abbreviations: achene diameter (ad); disease incidence (DI); dry root weight (DRW); drought score (DS); dry shoot weight (DSW); electrolyte leakage (EC); fresh root weight (FRW); fresh shoot weight (FSW); head diameter (HD); leaf area (LA); number of leaves (NL); plant height (PH); proline (PRO); relative water content (RWC); stem diameter(SD); total chlorophyll (TCHL).

Figure S7: PCA biplot of sunflower growth, agronomic, and physiological traits under mild drought stress (75% field capacity) with bacterial inoculations. PC1 and PC2 explained 72.79% and 19.01% of the total variance, respectively. Treatment groups (CTRL, M4, M34, and MC) exhibited distinct clustering patterns, indicating differential physiological and growth responses under mild water limitation. Trait abbreviations are described in Figure S6.

Figure S8: PCA biplot of sunflower responses under moderate drought stress (50% field capacity) with bacterial inoculations. PC1 (61.42%) and PC2 (25.20%) accounted for a substantial proportion of the total variance. The biplot illustrates clear treatment separation based on associations with growth and drought‐responsive traits, indicating varying adaptive responses to water deficit. Trait abbreviations are as defined in Figure S6.

Table S1: Cultural and biochemical characterization of P. protegens M4 and C. braakii M34.

Table S2: Genome‐based identification of functionally annotated genes related to nutrient acquisition, hormonal regulation, and stress tolerance in endophytic P. protegens strain M4.

Table S3: Genomic determinants of plant growth promotion and stress modulation in C. braakii strain M34.

Table S4: Predicted secondary metabolite biosynthetic gene clusters in P. protegens M4 genome.

Table S5: Predicted secondary metabolite gene clusters in the genome of C. braakii strain M34.

Table S6: Effect of bacterial treatments on the growth, agronomic, and physiological parameters of sunflower under100% water field capacity.

Table S7: Effect of bacterial treatments on the growth, agronomic, and physiological parameters of sunflower under 75% water field capacity.

Table S8: Effect of bacterial treatments on the growth, agronomic, and physiological parameters of sunflower under 50% water field capacity.

Table S9: Effect of bacterial treatments on the growth, agronomic, and physiological parameters of sunflowers under 25% water field capacity.

Table S10: Growth parameters of sunflower under different inoculation treatments and drought levels.

PLD3-10-e70184-s001.docx^{(764.9KB, docx)}

Acknowledgments

O.O.B. recognizes the National Research Foundation (NRF) South Africa for grants (UID123634 and UID132595) that support this research. M.M. expresses gratitude to North‐West University for bursary provisions. The graphic abstract was created with the help of https://BioRender.com.

Data Availability Statement

All data supporting the findings of this study are included in this article and its Supporting Information or are available through publicly accessible databases. The draft genome sequences of Pseudomonas protegens M4 and C. braakii M34 have been deposited in the NCBI database under accession numbers JBQRVA000000000 and JBQUYN000000000, respectively. The raw sequencing reads are available under BioSample accession numbers SAMN50614363 (M4) and SAMN50614364 (M34) within BioProject PRJNA1305676. Sequence reads have also been archived in the Sequence Read Archive (SRA) under accession numbers SRR35264040 (M4) and SRR35264041 (M34).

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