Functional Characterization of Wheat Seed Endophytic Bacteria Reveals Plant Growth-Promoting Traits and Potential Biocontrol Activity Against Fusarium Pathogens

Abstract

Seed-associated microbiota are emerging as key determinants of early plant establishment and resilience, yet their functional potential in wheat remains underexplored. Here, we isolated and functionally characterized culturable bacterial endophytes from seeds of Triticum aestivum cvs. Saekeumkang, Baeggang, and Ariheuk to assess plant growth promotion and pathogen suppression. Sixteen bacterial strains, belonging to Pseudomonadota, Actinomycetota, and Bacillota, were identified using 16S rRNA gene sequencing. Functional assays revealed the high prevalence of plant growth-promoting traits, with all isolates producing indole-3-acetic acid and 56.25% exhibiting phosphate-solubilizing activity, whereas siderophore production was restricted to Pseudomonas poae WSSR12. Despite this apparent functional redundancy, in planta assays demonstrated strong strain-specific effects on seedling biomass. Neobacillus cucumis WSSR17 consistently induced the highest increase in fresh weight. In parallel, dual culture assays against multiple Fusarium pathogens revealed that only one isolate, Calidifontibacillus erzurumensis WSSR11, showed consistent antifungal activity across all tested Fusarium strains. Notably, isolates combining multiple functional traits did not always correspond to the strongest growth promotion, underscoring the importance of host-microbe compatibility and trait expression in planta. Collectively, these findings reveal a functionally diverse seed endophytic community with complementary roles in plant growth and disease suppression. We propose that rational selection and combination of complementary strains, particularly N. cucumis WSSR17 (growth promotion), C. erzurumensis WSSR11 (biocontrol), and P. poae WSSR12 (multifunctional nutrient mobilization) could enable the development of targeted, multi-strain bioinoculant strategies for wheat. This study advances our understanding of seed microbiome functionality and provides a foundation for microbiome-informed crop improvement.

Wheat (Triticum aestivum L.) is one of the world’s most important staple crops, and improving its productivity in a sustainable manner remains a major agricultural priority (Chen et al., 2022). Seeds are increasingly recognized as important reservoirs of plant-associated microbes because they provide the earliest microbial inoculum for seedling establishment and early plant development (Shade et al., 2017). In wheat, seed-associated bacterial communities have been shown to vary with genotype and stress background, and several studies have identified beneficial taxa from wheat seeds, including members of Bacillus, Paenibacillus, Pantoea, Curtobacterium, and Arthrobacter (Aswini et al., 2023; Hone et al., 2021; Kuźniar et al., 2020).

Wheat seed endophytic bacteria are of particular interest because they may promote plant growth through mechanisms such as indole-3-acetic acid production, phosphate solubilization, and siderophore secretion, while some strains may also suppress fungal pathogens (Berg and Smalla, 2009; Compant et al., 2005; Díaz Herrera et al., 2016; Hardoim et al., 2008; Rosenblueth and Martínez-Romero, 2006; White et al., 2019). This multifunctional potential is especially relevant for wheat, in which beneficial seed-borne bacteria could support early seedling growth while also contributing to biological control of important diseases such as those caused by Fusarium spp. (Chen et al., 2022; Díaz Herrera et al., 2016).

However, the functional characterization of culturable wheat seed endophytes remains limited, particularly for strains that combine both plant growth-promoting and antifungal traits (Aswini et al., 2023; Kuźniar et al., 2020). Because some seed endophytes can be vertically transmitted, naturally compatible with seed-coating based delivery systems, and able to colonize plant tissues from the earliest stages of germination, they represent a particularly attractive microbial resource for developing practical bioinoculants in wheat.

Therefore, the aim of the present study was to isolate and characterize wheat seed endophytic bacteria and to evaluate their potential as plant growth promoters and biocontrol agents. Specifically, the objectives were to assess indole-3-acetic acid production, phosphate solubilization, and siderophore production, to determine antifungal activity against Fusarium pathogens, and to evaluate their effects on wheat seedling fresh weight.

Materials and Methods

Surface sterilization and disinfection of seeds

To isolate seed endophytes, wheat seeds were placed in sterile Falcon tubes containing sterile distilled water (SDW) and vortexed for 1 min to remove loosely adhered surface debris before surface sterilization and disinfection. The seeds were then submerged in 70% ethanol for 5 min, followed by 2.5% sodium hypochlorite for 3 min, to disinfect the seed surface. After disinfection, the seeds were thoroughly rinsed with SDW ten times to remove any residual disinfectants. To validate the effectiveness of the surface sterilization protocol, 100 μL of the final rinse water was spread onto the same culture media used for endophytic bacterial isolation. The plates were incubated at 30°C for 7 days. The absence of bacterial growth on these plates confirmed the efficacy of the surface sterilization procedure.

Isolation of seed endophytic bacteria

Surface-sterilized wheat seeds (3–4 per sample) were crushed using a sterile mortar and pestle and suspended in SDW. The suspension was spread onto ½ Reasoner’s 2A agar (R2A; yeast extract 0.25 g/L, proteose peptone No. 3 0.25 g/L, casamino acids 0.25 g/L, dextrose 0.25 g/L, soluble starch 0.25 g/L, sodium pyruvate 0.15 g/L, dipotassium phosphate 0.15 g/L, magnesium sulfate 0.025 g/L, agar 20 g/L). The cultured plates were incubated at 30°C for 2 weeks to allow for the growth of diverse endophytic bacteria. Following incubation, morphologically distinct bacterial colonies were selected and purified. Pure cultures of the isolated seed endophytes were suspended in 40% glycerol and stored at −80°C for further use.

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from pure bacterial isolates using the AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea) according to the manufacturer’s instructions. For each of the isolated bacterial strains, a single colony was picked, purified, and subjected to PCR amplification using the 16S rRNA gene with primers 27F/1492R (Lane, 1991). PCR reactions were performed in a total volume of 20 μL containing 3 μL of template DNA, 10 μL of GoTaq Green Master Mix (Promega, Madison, WI, USA), 5 μL of nuclease-free water, and 1 μL (1 μM) of each primer. The PCR conditions consisted of an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min and 30 s, with a final extension at 72°C for 5 min. To remove residual dNTPs and primers after PCR, 5 μL of the PCR product was treated with 2 μL of ExoSAP IT (Thermo Fisher Scientific Inc., Waltham, MA, USA) and incubated at 37°C for 15 min, followed by enzyme inactivation at 85°C for 15 min. DNA sequencing was conducted at the DNA sequencing facility of Macrogen (Seoul, Korea). The nucleotide sequence of the isolate was identified by comparing it with sequences in the NCBI database.

Screening for plant growth-promoting traits

The plant growth-promoting traits of wheat seed endophytic bacteria, including indole-3-acetic acid (IAA) production, siderophore production, and phosphate solubilization, were evaluated using standard assays.

Salkowski’s reagent test for indole-3-acetic acid detection

The ability of seed endophytes to produce IAA was determined using the Salkowski reagent (Gordon and Weber, 1951). All strains were initially pre-cultured on LB agar. For the IAA production assay, isolates that grew on LB were inoculated into LB medium supplemented with 0.15% (w/v) L-tryptophan and incubated at 30°C for 7 days. However, because strains WSSR1 and WSSR15 failed to grow on LB medium during pre-culturing, these strains were instead cultured in 1/2 R2B medium supplemented with the same concentration of L-tryptophan. Accordingly, the corresponding uninoculated control for these strains was also prepared using 1/2 R2B medium as the culture medium. Following incubation, the cultures were centrifuged at 12,300 g for 5 min to obtain cell-free supernatants. To detect IAA production, 1 mL of each bacterial supernatant was mixed with 2 mL of Salkowski reagent (2 mL of 0.5 M FeCl₃ in 98 mL of 35% HClO₄) in a test tube. The mixture was incubated in the dark at room temperature for 30 min. The development of a pink to red color (OD₅₃₆) indicated the presence of IAA in the bacterial supernatant.

Phosphate solubilization assay

The phosphate solubilizing ability of the endophytic bacteria was evaluated using a modified Pikovskaya medium (Nautiyal, 1999). The solid Pikovskaya medium was prepared using commercial Pikovskaya’s Broth (HiMedia, Mumbai, India), supplemented with 15 g/L agar to prepare the solid plates. The composition of the medium (g/L) was as follows: dextrose (10.0), Ca₃(PO₄)2 (5.0), (NH₄)₂SO₄ (0.5), KCl (0.2), MgSO₄ (0.1), MnSO₄ (0.0001), FeSO₄ (0.0001), and yeast extract (0.5), and distilled water (1,000 mL). The medium was autoclaved at 121°C for 15 min and allowed to cool before pouring into Petri dishes. A 10 μL aliquot of the bacterial suspension (OD₆₀₀ = 0.5) was drop-inoculated onto the center of the solidified Pikovskaya medium. The plates were then incubated at 30°C for 7 days under aerobic conditions. Following incubation, the plates were visually examined for clear zones around the bacterial colonies. For strains that formed opaque colonies, the colonies were carefully removed using an inoculation loop to clearly observe any underlying phosphate solubilization zones. The formation of clear zones surrounding the bacterial colonies was considered a positive result for phosphate solubilization. The absence of clear zones indicated the inability of the bacterial isolates to solubilize inorganic phosphate under the given experimental conditions.

Siderophore production assay

The chrome azurol S (CAS) agar assay, as described by Schwyn and Neilands (1987), was employed to evaluate the siderophore-producing ability of bacterial endophytes isolated from seeds. The CAS agar medium was prepared by adding 750 mL of deionized water to 100 mL of 10× MM9 salt solution, which contained 75.12 g/L sodium phosphate dibasic dihydrate, 0.9 g/L potassium phosphate, 5 g/L sodium chloride, 10 g/L ammonium chloride, and 15 g/L agar. PIPES (1,4-piperazinediethanesulfonic acid) were added at a concentration of 30.24 g/L, and the pH was adjusted to 6.8 using sodium hydroxide. The medium was then autoclaved at 121°C for 15 min. After cooling the medium to 50–55°C, 30 mL of filter-sterilized casamino acid solution (10%, w/v) and 10 mL of filter-sterilized glucose solution (20%, w/v) were aseptically added. The blue dye solution was prepared by combining a solution of 0.0605 g CAS dissolved in 50 mL of deionized water with an iron solution made by dissolving 0.0027 g of iron (III) chloride hexahydrate in 10 mM hydrochloric acid. HDTMA (hexadecyltrimethylammonium bromide) was added to 40 mL of deionized water at a concentration of 0.0729 g, and this solution was combined with the CAS-iron solution, followed by autoclaving at 121°C for 15 min (Louden et al., 2011; Milagres et al., 1999). The blue dye solution (100 mL) was carefully added to the CAS agar medium along the wall of the container while stirring, and the medium was then poured into Petri dishes. Seed endophytes were cultured in either tryptic soy broth or ½ R2B medium at 30°C with shaking at 160 rpm. For the CAS agar assay, 5 μL of each liquid-cultured seed endophyte was inoculated onto the CAS agar plates and incubated at 30°C in the dark for 7 days. Siderophore production was indicated by the formation of an orange halo around the bacterial colonies.

Plant growth promotion assay

The plant growth-promoting (PGP) effects of endophytic bacteria were evaluated using a soil-based seedling assay. Wheat seeds were surface sterilized prior to use. Seed endophytic bacterial strains were cultured in appropriate growth media and incubated until reaching the exponential phase. The bacterial cultures were then adjusted to an optical density (OD₆₀₀) of 1.0.

For the plant growth assay, 15 mL of each bacterial suspension was inoculated into 17 g of sterilized potting soil and mixed thoroughly to ensure uniform distribution. The inoculated soil was incubated for 24 h to allow bacterial establishment. Surface-sterilized seeds were then directly sown into the treated soil. Seedlings were grown under 16 h light (24°C) / 8 h dark (22°C), and plant growth-promoting effects were evaluated four weeks after sowing by measuring fresh weight of the seedlings.

Antifungal activity assay

The antifungal activity of seed endophytic bacteria was evaluated against multiple Fusarium pathogens using a dual culture method. The fungal pathogens tested included F. graminearum strains GZ03639, GWS 16-4-7, and GWR-12R5, as well as F. asiaticum (strain SCK04).

Seed endophytic bacterial strains were cultured for 3 days, and bacterial suspensions were prepared and adjusted to an optical density (OD₆₀₀) of 1.0. Fungal mycelial plugs were obtained from actively growing cultures and placed at the center of potato dextrose agar (PDA) plates. Paper disks inoculated with 50 μL of bacterial suspension were placed at an appropriate distance from the fungal plugs on the same plates. The co-inoculated plates were incubated at 25°C.

Fungal growth was monitored after 4–7 days of incubation, and inhibition of fungal growth was determined by comparing mycelial expansion in the presence and absence of bacterial isolates.

Statistical analysis

Statistical analyses were performed using the R programming environment (version 4.3.2; R Core Team, 2023). Because the fresh-weight data did not satisfy the assumption of normality (Shapiro-Wilk test, P < 0.05), non-parametric analyses were used. Fresh weight data were analyzed using the Kruskal–Wallis test to evaluate differences among treatments. When significant differences were detected, post hoc pairwise multiple comparisons were performed using a rank-based test with P-value adjustment. Statistical significance was considered at P < 0.05. Different lowercase letters indicate significant differences among treatments, whereas treatments sharing at least one letter are not significantly different.

Data deposition

All 16S rRNA gene sequences of the isolated endophytes in this study have been deposited in the NCBI GenBank database under accession numbers PZ403196-PZ403211. These sequences are accessible through the following link: http://www.ncbi.nlm.nih.gov/genbank

Results

Isolation and taxonomic identification of seed endophytic bacteria

A total of 16 bacterial endophytes were identified from wheat seeds using a culture-dependent approach (Table 1). Taxonomic identification based on 16S rRNA gene sequencing revealed that the isolates belonged to Pseudomonadota (43.8%), Actinomycetota (31.2%), and Bacillota (25.0%) at the phylum level (Fig. 1A). At the family level, the isolates were distributed across multiple families, with Sphingomonadaceae and Bacillaceae (18.8%), and Microbacteriaceae and Methylobacteriaceae (12.5%) among the most represented groups, followed by other families with lower proportions (Fig. 1B).

Table 1.

Identification of bacterial strains isolated from surface-sterilized seeds of wheat cultivars Saekeumkang, Baeggang, and Ariheuk

Strain	Cultivar	Culture medium	Taxon name	Similarity	Hit strain name
WSSR1	Saekeumkang	1/2 R2A	Methylobacterium terricola	99.09%	17Sr1-39
WSSR3	Saekeumkang	1/2 R2A	Sphingomonas roseiflava	100%	MK341
WSSR4	Saekeumkang	1/2 R2A	Massilia phosphatilytica	99.09%	12-OD1
WSSR5	Saekeumkang	1/2 R2A	Sphingomonas endophytica	99.92%	YIM 65583
WSSR6	Saekeumkang	1/2 R2A	Curtobacterium pusillum	99.27%	DSM 20527
WSSR7	Saekeumkang	1/2 R2A	Curtobacterium flaccumfaciens	99.77%	20TX0166
WSSR8	Saekeumkang	1/2 R2A	Paenibacillus barengoltzii	99.80%	SAFN-016
WSSR11	Saekeumkang	1/2 R2A	Calidifontibacillus erzurumensis	100.00%	P2
WSSR12	Saekeumkang	1/2 R2A	Pseudomonas poae	99.92%	P 527/13
WSSR14	Saekeumkang	1/2 R2A	Micrococcus yunnanensis	99.92%	YIM 65004
WSSR15	Saekeumkang	1/2 R2A	Methylorubrum extorquens	100%	IAM 12631
WSSR16	Saekeumkang	1/2 R2A	Sphingomonas endophytica	99.92%	YIM 65583
WSSR17	Saekeumkang	1/2 R2A	Neobacillus cucumis	99.55%	AP-6
WSSR18	Saekeumkang	1/2 R2A	Dermacoccus nishinomiyaensis	99.78%	DSM 20448
WSBR2	Baeggang	1/2 R2A	Bacillus safensis	100.00%	FO-36b
WSAR1	Ariheuk	1/2 R2A	Actinomadura montaniterrae	99.08%	CYP1-1B

Fig. 1.

Taxonomic distribution of wheat seed endophytic bacteria. (A) Distribution of bacterial isolates at the phylum level. (B) Distribution of bacterial isolates at the family level.

Production of IAA

All 16 bacterial isolates exhibited IAA production, as indicated by the development of pink to red coloration upon reaction with Salkowski’s reagent (Fig. 2).

Fig. 2.

Indole-3-acetic acid (IAA) production by endophytic bacterial strains isolated from wheat cultivars Saekeumkang, Baeggang, and Ariheuk seeds was detected using Salkowski’s reagent. The development of a pink-red color indicated the presence of IAA. The IAA-producing strains were identified based on the color change in the corresponding cuvettes.

Phosphate solubilizing ability of bacterial seed endophytes

Phosphate-solubilizing activity was assessed in wheat seed endophytic bacterial isolates using Pikovskaya’s agar (Fig. 3). Clear halo formation around bacterial colonies was considered positive for phosphate solubilization. Among the 16 tested isolates, nine strains exhibited phosphate-solubilizing activity: Bacillus safensis WSBR2, Sphingomonas roseiflava WSSR3, Massilia phosphatilytica WSSR4, Curtobacterium pusillum WSSR6, Curtobacterium flaccumfaciens WSSR7, Pseudomonas poae WSSR12, Sphingomonas endophytica WSSR16, Neobacillus cucumis WSSR17, and Dermacoccus nishinomiyaensis WSSR18. These positive isolates represented 56.25% of the total bacterial collection.

Fig. 3.

Assessment of phosphate-solubilizing ability of seed endophytic bacteria isolated from wheat cultivars Saekeumkang, Baeggang, and Ariheuk using Pikovskaya’s agar. The formation of clear halos around bacterial spots indicates phosphate solubilization by the endophytic strains. The inoculated bacterial strain is mentioned at the bottom of each plate. For certain strains that formed opaque or dense colonies, the bacterial mass was gently removed after incubation to clearly visualize the solubilization zones underneath.

Siderophore production by bacterial isolates using CAS agar assay

Only one bacterial strain (P. poae, WSSR12) exhibited siderophore production, as evidenced by the formation of an orange halo on CAS agar, while all other strains showed no detectable siderophore activity (Fig. 4).

Fig. 4.

Assessment of siderophore-producing ability of seed endophytic bacteria isolated from wheat cultivars Saekeumkang, Baeggang, and Ariheuk using the chrome azurol S agar assay. The formation of an orange halo around a bacterial colony indicates siderophore production. Strain identities are shown below the corresponding plates.

Growth-promoting effects of bacterial strains on wheat seedlings

The PGP potential of wheat seed endophytic bacteria was evaluated, focusing on their effects on seedling fresh weight (Fig. 5). The fresh weight of wheat seedlings varied significantly among treatments, as indicated by the Kruskal–Wallis test (P < 0.001). Among all bacterial treatments, Neobacillus cucumis WSSR17 produced the highest fresh weight and was grouped in statistical category “a”, indicating a significantly greater effect compared with several lower-performing treatments. Massilia phosphatilytica WSSR4 also showed relatively high fresh weight values and was classified as “ab”, suggesting performance comparable to N. cucumis WSSR17 but not significantly different from some intermediate groups.

Fig. 5.

Effect of different bacterial endophytic treatments on the fresh weight of wheat seedlings. Statistical differences among treatments were evaluated using the Kruskal–Wallis test, which showed a significant overall treatment effect (P < 0.001). Different lowercase letters above the boxplots indicate significant differences among treatments according to post hoc multiple-comparison analysis, whereas treatments sharing at least one letter are not significantly different from each other.

Intermediate fresh weight responses were observed for Methylobacterium terricola WSSR1, Sphingomonas endophytica WSSR5, Calidifontibacillus erzurumensis WSSR11, Pseudomonas poae WSSR12, Dermacoccus nishinomiyaensis WSSR18, and Methylorubrum extorquens WSSR15, all of which were assigned to overlapping statistical groups (“abc”), indicating no clear statistical separation among these treatments. Curtobacterium flaccumfaciens WSSR7 and Sphingomonas endophytica WSSR16 were grouped as “bc”, showing moderate effects that were not significantly different from the control or some intermediate treatments.

The lowest fresh weight values were recorded for Micrococcus yunnanensis WSSR14 and Bacillus safensis WSBR2 (group “c”); these isolates produced significantly lower fresh weight than Neobacillus cucumis WSSR17 (group “a”) but were not significantly different from the uninoculated control (group “bc”). Overall, only N. cucumis WSSR17 and Massilia phosphatilytica WSSR4 produced fresh weight values that were significantly higher than those of the lowest-performing groups, while all other treatments were statistically indistinguishable from the uninoculated control.

Antifungal activity of wheat seed endophytic bacteria against Fusarium pathogens

The antifungal activity of the 16 wheat seed endophytic bacterial isolates was evaluated against four Fusarium pathogens (F. graminearum strains GZ03639, GWS 16-4-7, and GWR-12R5, and F. asiaticum strain SCK04) using a dual culture assay. Of the 16 isolates tested, only Calidifontibacillus erzurumensis WSSR11 exhibited antifungal activity against the four Fusarium pathogens, whereas the remaining isolates showed no detectable inhibition; representative interactions between WSSR11 and each pathogen are shown in Fig. 6A–6D.

Fig. 6.

Antifungal activity of wheat seed endophytic bacterium Calidifontibacillus erzurumensis WSSR11 evaluated using a dual-culture assay. Representative interactions between WSSR11 and (A) Fusarium graminearum strain GZ03639, (B) F. graminearum strain GWS 16-4-7, (C) F. graminearum strain GWR-12R5, and (D) F. asiaticum strain SCK04 are shown.

Discussion

The present study demonstrates that wheat seeds harbor culturable bacterial endophytes with diverse functional traits related to plant growth promotion and biocontrol. Seed-borne endophytes are particularly attractive microbial resources for agricultural applications because of their natural compatibility with seed-based microbial delivery systems. Unlike externally introduced rhizosphere microorganisms, seed endophytes are already adapted to survival within seed tissues and may establish during the earliest stages of germination and seedling development (Shahzad et al., 2018; Truyens et al., 2015). Their natural association with seeds makes them promising candidates for seed-coating formulations and bioinoculant technologies, particularly because early colonization, persistence within plant tissues, and timely establishment during seedling development are important factors influencing microbial inoculant performance (Truyens et al., 2015; Verma et al., 2019). In addition, vertically transmitted seed endophytes may possess competitive advantages during early microbiome assembly because they colonize internal plant tissues before extensive recruitment of soil-derived microorganisms occurs (Shade et al., 2017; Verma et al., 2019). Therefore, wheat seed endophytes represent not only an ecologically important component of the plant microbiome but also a practical microbial resource for the future development of sustainable seed-treatment and crop-improvement strategies.

The taxonomic composition observed (Fig. 1A) is consistent with previous studies showing that seed endophytes commonly include Pseudomonadota, Actinomycetota, and Bacillota (Afzal et al., 2019; Truyens et al., 2015). Several genera detected in this study (Table 1), including Sphingomonas, Methylobacterium, Pseudomonas, Massilia, Curtobacterium, Micrococcus, Bacillus, Paenibacillus, and Actinomadura have also been reported from seed-associated bacterial communities in different crops, including wheat and other Poaceae species (Aswini et al., 2023; Verma and White, 2018). This supports the idea that wheat seeds maintain a functionally relevant culturable endophytic fraction that may contribute to early seedling performance.

Several genera recovered in the present study correspond to taxa previously assigned important ecological positions in wheat-associated microbial networks. For example, Sphingomonas has been identified among hub taxa in dryland wheat rhizosphere networks, while Massilia and Pseudomonas have been proposed as potentially important or keystone genera in wheat phyllosphere communities (Li et al., 2023; Schlatter et al., 2020). Accordingly, the recovery of Sphingomonas, Massilia, and Pseudomonas isolates from wheat seeds suggests possible taxonomic overlap between the present culturable collection and ecologically influential members of previously characterized wheat-associated microbial communities. Nevertheless, hub and keystone status is dependent on plant compartment, host genotype, environmental conditions, taxonomic resolution, and network-construction method. Therefore, the isolates identified here cannot be designated as hub or keystone taxa without community-level network analysis and experimental validation. Future studies combining culture-dependent isolation with amplicon sequencing, co-occurrence network analysis, and targeted community perturbation could determine whether these strains occupy central ecological positions in the wheat microbiome.

Neobacillus cucumis WSSR17 showed the strongest growth-promoting effect, producing the only fresh-weight increase that was statistically significant relative to the uninoculated control (group “a”; Fig. 5). Massilia phosphatilytica WSSR4 also produced relatively high fresh weight (group “ab”) but, sharing a letter with the control, was not significantly different from it, and the remaining isolates were statistically indistinguishable from the uninoculated treatment (Fig. 5, Table 2). Such strain-level variation is expected because endophytic bacteria differ in their ability to produce phytohormones, mobilize nutrients, colonize seedlings, and interact with host physiology (Afzal et al., 2019; Compant et al., 2019). Similar strain-dependent growth promotion has been reported for seed-derived endophytes in wheat and other cereals, where some isolates enhanced seedling development while others showed weak or neutral effects (Díaz Herrera et al., 2016; Kumar et al., 2021; Verma and White, 2018).

Table 2.

Plant growth-promoting properties of bacterial endophytes isolated from surface-sterilized seeds of wheat cultivars Saekeumkang, Baeggang, and Ariheuk

Strain	Taxon name	Production of IAA	Phosphate solubilization	Antifungal activity	Production of Siderophores	Plant growth promoting
WSSR1	Methylobacterium terricola	+	−	−	−	−
WSSR3	Sphingomonas roseiflava	+	+	−	−	N/A
WSSR4	Massilia phosphatilytica	+	+	−	−	−
WSSR5	Sphingomonas endophytica	+	−	−	−	−
WSSR6	Curtobacterium pusillum	+	+	−	−	N/A
WSSR7	Curtobacterium flaccumfaciens	+	+	−	−	−
WSSR8	Paenibacillus barengoltzii	+	−	−	−	N/A
WSSR11	Calidifontibacillus erzurumensis	+	−	+	−	−
WSSR12	Pseudomonas poae	+	+	−	+	−
WSSR14	Micrococcus yunnanensis	+	−	−	−	−
WSSR15	Methylorubrum extorquens	+	−	−	−	−
WSSR16	Sphingomonas endophytica	+	+	−	−	−
WSSR17	Neobacillus cucumis	+	+	−	−	+
WSSR18	Dermacoccus nishinomiyaensis	+	+	−	−	−
WSBR2	Bacillus safensis	+	+	−	−	−
WSAR1	Actinomadura montaniterrae	+	−	N/A	−	N/A

+, Biological activity detected; −, not detected; N/A, not applicable.

IAA production (Fig. 2) and phosphate solubilization (Fig. 3) were among the main growth-promoting traits detected in the present study. IAA-producing endophytes can influence root architecture, lateral root development, and nutrient uptake, whereas phosphate-solubilizing bacteria can improve phosphorus availability by mobilizing insoluble phosphate sources (Duca et al., 2014; Mehta and Nautiyal, 2001; Pikovskaya, 1948). In this study, 16 isolates produced IAA (Fig. 2) and nine isolates solubilized phosphate (Fig. 3), indicating that a substantial proportion of wheat seed endophytes possessed nutrient- or hormone-related PGP traits (Table 2). However, the relationship between in vitro PGP traits and seedling fresh weight was not always direct. For example, Pseudomonas poae WSSR12 combined IAA production, phosphate solubilization, and siderophore production, but it was not among the strongest fresh-weight-promoting isolates. This suggests that plant growth promotion depends not only on possession of individual traits but also on trait expression in planta, colonization ability, and compatibility with the host genotype.

The distribution of these functional traits was broadly comparable to, although not identical with, those reported for other culturable wheat seed-endophyte collections. In a recent survey of wheat seed endophytes obtained from cultivars grown across different agroecological zones, approximately 89% of the tested isolates produced IAA, 78% solubilized phosphate, and 11% produced siderophores (Prasad et al., 2025). In comparison, all isolates in the present collection produced IAA, 56.25% solubilized phosphate, and 6.25% produced siderophores. These results suggest a high prevalence of auxin-related potential, an intermediate representation of phosphate-mobilizing activity, and relatively restricted siderophore production within the present collection. However, direct numerical comparisons among studies should be interpreted cautiously because functional frequencies are influenced by host genotype, isolation medium, precursor supplementation, incubation conditions, assay sensitivity, and the criteria used to define a positive response.

Siderophore production was detected only in P. poae WSSR12 (Fig. 4), making this isolate functionally distinct from the rest of the collection. Although siderophores can promote plant growth by improving iron acquisition and may contribute to biocontrol by restricting iron availability to fungal pathogens (Schwyn and Neilands, 1987), these functions were not reflected in our results, as WSSR12 neither produced the strongest biomass response nor exhibited antifungal activity. Notably, the only siderophore-producing isolate (WSSR12) and the only antifungal isolate (C. erzurumensis WSSR11) did not coincide, suggesting that the antifungal activity of WSSR11 is mediated by mechanisms other than siderophore-based iron competition. WSSR12 is therefore best regarded as a multifunctional nutrient-mobilizing candidate that requires further validation under greenhouse and pathogen-challenge conditions.

C. erzurumensis WSSR11 showed the most consistent antifungal activity across the tested Fusarium pathogens, suggesting that this isolate is the strongest biocontrol candidate in the present collection (Table 2). In contrast, all other tested isolates showed no antifungal activity against the tested Fusarium pathogens under the present assay conditions. This pattern agrees with previous studies showing that wheat seed endophytes can suppress F. graminearum, but that antagonistic activity varies strongly by bacterial strain and fungal isolate (Díaz Herrera et al., 2016; Gdanetz and Trail, 2017). Endophytic biocontrol mechanisms may include antibiosis, competition for nutrients or space, siderophore-mediated iron competition, hydrolytic enzyme activity, and induction of host defense responses (Chaudhary et al., 2022; Morales-Cedeño et al., 2021; Pliego et al., 2011). As a member of the Bacillaceae, WSSR11 may rely on mechanisms commonly reported within this family, such as the secretion of antifungal lipopeptides, hydrolytic enzymes, and volatile organic compounds, although the specific basis of its anti-Fusarium activity remains to be elucidated.

An important practical implication of the present study is that the beneficial functions identified were distributed across different bacterial isolates rather than concentrated within a single strain. N. cucumis WSSR17 showed the strongest plant growth-promoting effect based on seedling fresh weight (Fig. 5), whereas C. erzurumensis WSSR11 exhibited the most consistent antifungal activity against multiple Fusarium pathogens (Fig. 6A–6D). In addition, P. poae WSSR12 exhibited multiple plant growth-promoting traits, including IAA production, phosphate solubilization, and siderophore production, although its effect on seedling fresh biomass was intermediate rather than among the highest-performing isolates (Table 2). These findings demonstrate that possession of multiple in vitro functional traits does not necessarily translate directly into superior plant growth promotion under in planta conditions. Instead, the results suggest that bacterial performance depends on a combination of factors including host compatibility, colonization efficiency, metabolic activity within plant tissues, and ecological interactions with other microorganisms. Future studies should therefore evaluate the establishment and persistence of individual strains within wheat tissues using strain-specific quantitative PCR, tagged-strain imaging, or amplicon-based microbial community profiling. Examining the in planta expression of microbial IAA-biosynthesis genes and quantifying IAA or related indole metabolites within colonized seedlings would further help determine whether the discrepancy between in vitro IAA production and plant growth promotion results from differences in colonization efficiency, metabolic activity, or host responsiveness.

Collectively, the observed functional complementarity among isolates suggests that single-strain inoculation strategies may be insufficient for simultaneously maximizing plant growth promotion and pathogen suppression. Rather, the combination of functionally distinct isolates into a multi-strain consortium may represent a more effective approach for achieving stable and multi-faceted benefits in wheat production. For example, a consortium combining the strong biomass-promoting effects of N. cucumis WSSR17, the consistent antifungal activity of C. erzurumensis WSSR11, and the multifunctional nutrient-mobilizing traits of P. poae WSSR12 could potentially provide complementary benefits that are not achievable through individual strains alone. Similar advantages of consortium-based microbial inoculants have been reported in other plant systems, where functional diversity among strains improves colonization stability, nutrient acquisition, stress tolerance, and disease suppression (Adeleke et al., 2021; Compant et al., 2005; Morales-Cedeño et al., 2021). Therefore, the present findings support the development of rationally designed seed-endophyte consortia as a promising strategy for future wheat bioinoculant development.