Characterization of Tomato Seed Endophytic Bacteria as Growth Promoters and Potential Biocontrol Agents

Mehwish Roy; Byeonghyeok Kang; Seongeun Yang; Heeyun Choi; Kihyuck Choi

doi:10.5423/PPJ.OA.09.2024.0142

Plant Pathol J > Volume 40(6); 2024 > Article

Roy, Kang, Yang, Choi, and Choi: Characterization of Tomato Seed Endophytic Bacteria as Growth Promoters and Potential Biocontrol Agents

Research Article

The Plant Pathology Journal 2024;40(6):578-592.

Published online: December 1, 2024

DOI: https://doi.org/10.5423/PPJ.OA.09.2024.0142

Characterization of Tomato Seed Endophytic Bacteria as Growth Promoters and Potential Biocontrol Agents

Mehwish Roy^†, Byeonghyeok Kang^†, Seongeun Yang, Heeyun Choi, Kihyuck Choi^*

Department of Applied Bioscience, Dong-A University, Busan 49315, Korea

^*Corresponding author. Phone) +82-51-200-7586, FAX) +82-51-200-7505, E-mail) hyuck1857@dau.ac.kr

^† These authors contributed equally to this work.

Handling Editor: Hyun Gi Kong

(Received on September 11, 2024; Accepted on October 1, 2024)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Endophytic bacteria residing within plant seeds are increasingly recognized for their potential to enhance plant growth and provide biocontrol against pathogens. Despite this, seed-borne endophytes remain underexplored in many crops, including tomato. In this study, we isolated and characterized bacterial endophytes from tomato seeds and evaluated their plant growth-promoting traits and antifungal activities. The taxonomic analysis of the Hawaii 7996 tomato seed endophyte collection revealed a diverse community, predominantly from the phylum Bacillota, with Paenibacillaceae and Bacillaceae as the most abundant families. Among the 35 unique strains identified, 19 produced indole-3-acetic acid, four exhibited siderophore production, and 12 could solubilize phosphate. These traits contribute to growth promotion and disease suppression in plants. In the plant growth promotion assay, several bacterial strains, notably Streptomyces olivaceus (BHM1), Streptomyces variegatus (BHM3), Bacillus stercoris (BHR2), and Moraxella osloensis (YHT4-1), demonstrated significant potential for tomato cultivation by positively affecting fresh weight, stem length, and root length. These strains consistently promoted growth across all three parameters evaluated in this study. Furthermore, several strains exhibited strong antifungal activity against major tomato pathogens, including Fusarium oxysporum race 1 and 2, and Botrytis cinerea. Notably, Bacillus subtilis (BHN1), Bacillus stercoris (BHR2), and Paenibacillus peoriae (YHR2-1) showed broad-spectrum antifungal efficacy. Our findings highlight the potential of seed-associated endophytic bacteria as growth promoters and biological control agents, offering promising avenues for sustainable agricultural practices.

Keywords: antifungal activity, biological control agents, plant growth-promoting bacteria (PGPB), tomato seed endophytes

To reduce the negative impacts of fertilizers and chemical pesticides, alternative strategies for promoting plant growth and controlling fungal diseases are crucial. Endophytes within plant tissues have emerged as a promising approach to enhance crop growth and provide biological control against fungal pathogens (Zhu et al., 2020). These endophytes, primarily bacteria and fungi, colonize nearly all plant tissues, including roots, stems, leaves, fruits, and seeds, supporting plant development and adaptation to various environmental conditions (Glassner et al., 2018; Llorens et al., 2019; Márquez et al., 2007; White et al., 2019). The interactions between seed endophytes and their host plants have recently gained attention, particularly in sustainable agriculture (Misganaw et al., 2019).

Seed-associated endophytic bacteria are increasingly recognized for their ability to enhance germination processes (Pitzschke, 2016) and promote plant growth by producing hormones such as auxin and ethylene, mobilizing essential nutrients like nitrogen, phosphorus, and potassium, and synthesizing siderophores (Chang et al., 2021; Maheshwari et al., 2019; Ruiza et al., 2011; Soldan et al., 2019; Verma and White, 2018). As a crucial component of the plant microbiome, the seed microbiome represents both the endpoint of microbial community assembly in seeds and the starting point for the assembly of the microbiome in new seedlings (Shade et al., 2017). Seeds are vital plant structures that facilitate reproduction over time and enhance survival under stressful conditions, making them crucial in agriculture (Guan et al., 2009). Seeds harbor bacterial endophytes (López et al., 2018) that play a critical role in managing plant health and diseases by reducing pathogen population densities without eliciting hypersensitive reactions in the host (Hazarika et al., 2019; Roy et al., 2017).

Many endophytic bacteria, particularly Bacillus species, exhibit antagonistic activity against fungal pathogens by producing antimicrobial compounds. For instance, Bacillus species are known to generate antifungal lipopeptides, such as iturins, fengycins, surfactins, and bacillomycin. Bacillus subtilis SCB-1 has demonstrated antifungal activity against various fungal pathogens, including Alternaria and Fusarium species (Hazarika et al., 2019). Similarly, many studies have investigated the potential of B. stercoris for biological control. For example, B. stercoris A053, B. stercoris JNUCC, and B. stercoris X2 showed strong antifungal activity against plant pathogens (Byun et al., 2020; Dhruw et al., 2020; Guo et al., 2015).

Tomato (Solanum lycopersicum L.) is a widely cultivated vegetable crop known for its high nutritional content. However, it is highly susceptible to various fungal diseases, which can severely limit production (Panno et al., 2021). The succulent nature of its fruit increases its vulnerability to fungal infections. Major fungal pathogens affecting tomato include Rhizoctonia solani, Fusarium solani, Botrytis cinerea, Alternaria solani, and Verticillium species. These soil-borne pathogens are difficult to control due to their diverse host range and persistence in soil (Lamichhane et al., 2017). Although synthetic fungicides have been used to manage these diseases, their overuse has led to fungicide resistance and reduced biological control, highlighting the need for alternative, sustainable approaches (Soylu et al., 2010).

The objectives of this study were to isolate and identify endophytic bacterial strains from tomato seeds; evaluate the in vitro antagonistic activity of isolated strains against selected tomato fungal pathogens; assess the plant growth-promoting (PGP) traits of potential antagonistic strains; and investigate the efficacy of selected strains in tomato plants under controlled conditions. By exploring seed-derived endophytic bacteria as potential biological control agents and plant growth promoters, this research aims to contribute to the development of sustainable tomato cultivation practices.

Materials and Methods

Surface sterilization and disinfection of seeds

To isolate seed endophytes, tomato seeds were surface-sterilized and disinfected using the following procedure: Seeds were placed in a sterile microcentrifuge tube and vortexed for 1 min to remove any loosely adhered debris. 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 sterile distilled water (SDW) five 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 tomato seeds (3-4 per sample) were crushed using a sterile mortar and pestle and suspended in SDW. The suspension was then serially diluted to 10⁻¹ and 10⁻², and 100 μl of each dilution was spread onto various culture media, including tryptic soy agar (TSA; casein pancreatic digest 8.5 g/l, papaic digest of soybean 1.5 g/l, dextrose 1 g/l, sodium chloride 2.5 g/l, dipotassium phosphate 1.25 g/l, agar 15 g/l), Reasoner's 2A agar (R2A; yeast extract 0.5 g/l, proteose peptone no. 3 0.5 g/l, casamino acids 0.5 g/l, dextrose 0.5 g/l, soluble starch 0.5 g/l, sodium pyruvate 0.3 g/l, dipotassium phosphate 0.3 g/l, magnesium sulfate 0.05 g/l, agar 20 g/l), lysogeny agar (LB; casein enzymic hydrolysate 10 g/l, yeast extract 5 g/l, sodium chloride 10 g/l, agar 15 g/l), nutrient agar (NA; beef extract 3 g/l, peptone 5 g/l, agar 15 g/l), King's B medium (proteose peptone 20 g/l, dipotassium phosphate 1.5 g/l, magnesium sulphate heptahydrate 1.5 g/l, agar 15 g/l), V8 juice agar (V8; 200 ml/l, calcium carbonate 2 g/l, agar 20 g/l), and M9 salt agar (M9; disodium hydrogen phosphate 12.8 g/l, potassium dihydrogen phosphate 3.0 g/l, sodium chloride 10.5 g/l, ammonium chloride 1.0 g/l, casein enzymic hydrolysate 10 g/l, yeast extract 5 g/l, agar 15 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 by subculturing on their respective isolation media. 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 8F/1492R (Lane, 1991). PCR reactions were performed in a total volume of 20 μl containing 3 μl of template DNA, 10 μl of GoTaq polymerase (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 PGP traits

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

The ability of seed endophytes to produce indole-3-acetic acid (IAA) was determined using the Salkowski reagent (Gordon and Weber, 1951). Seed endophyte isolates were inoculated into LB medium supplemented with 0.15% (w/v) L-tryptophan and incubated at 30°C for 7 days. Following incubation, the cultures were centrifuged at 13,000 rpm 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 red color (OD₅₃₆) indicated the presence of IAA in the bacterial supernatant.

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 dehydrate, 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) was added at a concentration of 30.24 g/l, and the pH was adjusted to 7.0 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 R2B medium at 30°C with shaking at 120 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.

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 with the following composition (g/l): glucose (10.0), (NH₄)₂SO₄ (0.5), Ca₃(PO₄)₂ (5.0), NaCl (0.3), MgSO₄·7H₂O (0.3), KCl (0.2), MnSO₄·4H₂O (0.03), FeSO₄·7H₂O (0.03), yeast extract (0.5), agar (15.0), 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. The endophytic bacterial isolates were streaked in a straight line onto the solidified Pikovskaya medium using a sterile inoculation loop. The inoculated 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, indicating the solubilization of inorganic phosphate in the medium. 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.

Plant growth promotion assay

The PGP effects of seed endobacteria were evaluated using a hydroponic system. Surface-sterilized seeds were placed on sterile filter paper (ADVANTEC, Tokyo, Japan) in Petri dishes. Each dish was moistened with 5 ml of SDW and sealed. Seeds were incubated for germination under controlled conditions of 14 h light/10 h darkness at 28°C and 65% humidity for 1 week. Bacterial isolates were cultured on solid media specific to each strain for 3 days (Table 1). Colonies were then suspended in SDW to an optical density (OD₆₀₀) of 0.2. For the control group, an equivalent volume of SDW was used. Germinated seedlings were transferred to 5 ml tubes containing 5 ml of bacterial suspension. Care was taken to ensure that the cotyledons remained above the suspension level. Tubes were sealed, leaving a small opening for stem growth. The inoculated seedlings were grown under the same conditions as during germination (14/10 h light/dark cycle, 28°C, 65% humidity) for 2 weeks. After the 2-week growth period, PGP effects were evaluated by measuring fresh weight, stem length, and root length of the seedlings.

Antifungal activity assay

The antifungal activity of seed endophytes against plant pathogenic fungi was evaluated using the dual culture method. The fungal pathogens tested included Fusarium oxysporum f. sp. lycopersici race 1 and race 2 (causative agents of Fusarium wilt disease), R. solani (causing damping-off) and B. cinerea (causing gray mold disease). Seed endophyte cultures were grown for 3 days, and bacterial suspensions were prepared and adjusted to an optical density (OD₆₀₀) of 1.0. Fungal mycelial plugs, obtained from 7-day-old cultures using a cork borer, were placed on potato dextrose agar (PDA) medium. Paper disks inoculated with 50 μl of the bacterial suspensions were placed 4 cm away from the fungal plugs on the same PDA plates. The co-inoculated plates were incubated at 25°C. Fungal growth was assessed after 4 to 7 days of incubation, when the mycelia in the control plates (without endophytes) had fully colonized the Petri dish. The extent of growth inhibition was determined by comparing the growth of the pathogenic fungi in the presence and absence of the seed endophytes.

Statistical analysis

Statistical analyses were performed using the R programming environment to evaluate the PGP effects of seed endophytes. The appropriate statistical test including Welch's t-test, Student’s t-test, or Wilcoxon rank-sum test was applied based on the results of the Shapiro-Wilk and Levene tests for normality and homogeneity of variance (Gastwirth et al., 2019).

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 PQ285164 to PQ285198. 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 59 bacterial endophytes were initially isolated from the surface-disinfected seeds of Hawaii 7996 using a culture-dependent technique. After sequencing and comparison, 35 unique bacterial strains were identified (Table 1). The taxonomic composition of the seed endophytic bacterial community revealed a predominance of the phylum Bacillota, accounting for 79.7% of the identified species. Actinomycetota and Pseudomonadota represented the next most abundant phyla at 18.6% and 1.7%, respectively. At the family level, Paenibacillaceae (39%) and Bacillaceae (35.6%) were the most prevalent, followed by Staphylococcaceae (5.1%), Mycobacteriaceae (6.8%), Streptomycetaceae (5.1%), Micrococcaceae (3.4%), Nocardioidaceae (3.4%), and Moraxellaceae (1.7%). This taxonomic profile highlights the dominance of Bacillota and the diverse representation of other phyla and families within the seed endophytic bacterial community (Fig. 1A and B).

Screening of PGP traits of seed endophytic bacteria

The bacterial strains isolated from the Hawaii 7996 seeds were screened for PGP traits (Table 2).

Production of IAA

Salkowski's reagent was used to screen 35 bacterial strains for their ability to produce IAA, a key plant growth hormone. Nineteen bacterial strains, representing 13 different genera, exhibited positive results, as indicated by the development of an orange-red color. The strains belonged to the following species: Nocardioides marinus (10HR4), Peribacillus frigoritolerans (10HT1), Brevibacillus limnophilus (BHL1), Streptomyces olivaceus (BHM1), Streptomyces variegatus (BHM3), Staphylococcus capitis subsp. capitis (HSR_2), Micrococcus luteus (HSR_6), Bacillus acidicola (HSR_8), Bacillus pseudomycoides (HV17), Priestia megaterium (YHK1), Metabacillus idriensis (YHL1), Staphylococcus epidermidis (YHL2), Brevibacterium frigoritolerans (YHN1-1), Micrococcus yunnanensis (YHR1-1), Moraxella osloensis (YHT4-1), Paenibacillus glucanolyticus (YHT5-1), Paenibacillus cineris (YHTT1), Neobacillus cucumis (YHTT3), and Priestia aryabhattai (YNS1-2). The remaining 16 strains did not show evidence of IAA production under the tested conditions (Table 2, Fig. 2).

Production of siderophores

The CAS agar assay was used to evaluate the siderophore-producing ability of the seed endophytes. Four strains, identified as Mycolicibacterium mucogenicum (10HR1), Mycolicibacterium smegmatis (10HR3), S. variegatus (BHM3), and Paenibacillus peoriae (YHR2-1), demonstrated siderophore production, as evidenced by color change in the corresponding wells (Table 2, Fig. 3).

Phosphate solubilizing ability of bacterial seed endophytes

Phosphate solubilization ability was assessed using Pikovskaya's modified agar medium. Twelve endophytic strains, including representatives from M. smegmatis (10HR3), S. variegatus (BHM3), B. subtilis (BHN1), B. stercoris (BHR2), Fictibacillus enclensis (BHT1), Bacillus haynesii (BHT2), Staphylococcus warneri (HSR_1), S. capitis subsp. capitis (HSR_2), Cohnella suwonensis (HSR_3), P. megaterium (YHK1), S. epidermidis (YHL2), P. peoriae (YHR2-1), formed clear zones around their respective colonies, indicating their capacity to solubilize inorganic phosphate (Table 2, Fig. 4).

Growth-promoting effects of bacterial strains on tomato (Ye-gwang)

The PGP potential of isolated bacterial strains was evaluated on tomato cultivar Ye-gwang, focusing on their effects on fresh weight, stem length and root length (Table 2, Fig. 5). Eight bacterial strains significantly increased the fresh weight of tomato plants compared to the control (P < 0.001). These strains included: S. olivaceus (BHM1), B. stercoris (BHR2), M. luteus (HSR_6), B. acidicola (HSR_8), P. megaterium (YHK1), Paenibacillus barengoltzii (YHT3-1), M. osloensis (YHT4-1), and P. glucanolyticus (YHT5-1). Additionally, P. frigoritolerans (10HT1) and S. variegatus (BHM3) also led to significant increase in fresh weight compared to the control (P < 0.01). Five strains, namely B. subtilis (BHN1), S. capitis subsp. capitis (HSR_2), C. suwonensis (HSR_3), B. pseudomycoides (HV17), and Paenibacillus provencensis (YNS2-1) resulted in significant increase in fresh weight compared to the control (P < 0.05) (Table 2, Fig. 5A). For stem length, the bacterial strains that showed the most significant increase (P < 0.001) compared to the control were S. variegatus (BHM3), Cohnella massiliensis (BHN2), and B. stercoris (BHR2). Two strains, M. osloensis (YHT4-1) and P. provencensis (YNS2-1), also significantly increased stem length (P < 0.01). Additionally, six strains led to a significant increase in stem length compared to the control (P < 0.05): P. frigoritolerans (10HT1), B. limnophilus (BHL1), S. olivaceus (BHM1), B. subtilis (BHN1), F. enclensis (BHT1), and S. warneri (HSR_1) (Table 2, Fig. 5B). Regarding root length, S. variegatus (BHM3) and M. osloensis (YHT4-1) exhibited a highly significant increase in root length compared to control (P < 0.001). Additionally, significant increases in root length (P < 0.01) were observed for six strains: B. stercoris (BHR2), M. luteus (HSR_6), B. acidicola (HSR_8), B. frigoritolerans (YHN1-1), P. barengoltzii (YHT3-1), and P. glucanolyticus (YHT5-1). Five other strains, namely S. olivaceus (BHM1), B. subtilis (BHN1), S. capitis subsp. capitis (HSR_2), C. suwonensis (HSR_3), and M. yunnanensis (YHR1-1), demonstrated an increase in root length compared to the control (P < 0.05) (Table 2, Fig. 5C).

Antifungal activity of the bacterial seed endophytes against tomato plant pathogenic fungi

All the bacterial endophytes isolated from the Hawaii 7996 seeds were screened for their antifungal activity against four economically important fungal pathogens of tomato crops: F. oxysporum race 1, F. oxysporum race 2, R. solani and B. cinerea through a dual culture assay (Table 3).

Antifungal activity against F. oxysporum

For race 1, B. subtilis (BHN1), B. stercoris (BHR2), and P. peoriae (YHR2-1) demonstrated the strongest antagonistic activity (Fig. 6A). Although M. idriensis (YHL1), P. glucanolyticus (YHT5-1), and N. cucumis (YHTT3) are not included in Fig. 6A and did not exhibit antifungal activity as strong as that of the three aforementioned strains, they still demonstrated relatively weaker zones of inhibition on the medium (Table 3). Similarly, for race 2, B. subtilis (BHN1), B. stercoris (BHR2), and P. peoriae (YHR2-1) again exhibited the most pronounced antagonistic effects (Fig. 6B). Although P. barengoltzii (YHT3-1) and M. osloensis (YHT4-1) are not included in Fig. 6B and did not show as strong antifungal activity as the three aforementioned strains, they still demonstrated relatively weaker antifungal activity on the medium, as indicated by the formation of smaller inhibition zones around their respective colonies (Table 3).

Antifungal activity against R. solani

Among the 35 bacterial strains evaluated, B. subtilis (BHN1) and B. stercoris (BHR2) were the only two that exhibited antagonistic activity against the phytopathogenic fungus R. solani. However, the zones of inhibition produced by these strains were relatively weaker. Despite this reduced efficacy, the observed inhibition of R. solani growth by B. subtilis (BHN1) and B. stercoris (BHR2) suggests their potential as biocontrol agents (Table 3).

Antifungal activity against B. cinerea

Three stains, B. subtilis (BHN1), B. stercoris (BHR2), and P. peoriae (YHR2-1) showed the strongest antagonistic activity against B. cinerea (Fig. 6C). Although S. variegatus (BHM3), S. warneri (HSR_1), P. megaterium (YHK1), and P. aryabhattai (YNS1-2) are not included in Fig. 6C and did not demonstrate antifungal activity as strong as the three aforementioned strains, they still exhibited relatively weaker zones of inhibition around their corresponding colonies, indicating their ability to suppress the growth of B. cinerea (Table 3).

Discussion

Seed endophytes are crucial as beneficial microbes by enhancing plant growth, improving stress tolerance, and offering protection against pathogens. In this study, several endophytic bacterial strains exhibited important PGP activities. Notably, S. variegatus (BHM3) demonstrated a combination of key traits: production of IAA, phosphate solubilization, and siderophore production (Figs. 2-4). These traits collectively suggest a strong potential for BHM3 as a biofertilizer. Previous research has shown that certain phosphate-solubilizing bacteria also produce IAA, which not only enhances plant growth but improves phosphorus availability in soils (Barea et al., 1976; Khiangte and Lalfakzuala, 2021; Wan et al., 2020). The dual functionality of IAA production and phosphate solubilization makes BHM3 particularly valuable for promoting root development and increasing phosphorus bioavailability. Additionally, siderophore production contributes to improved iron acquisition by plants, further enhancing plant health and growth. The combination of these three PGP traits in S. variegatus (BHM3) makes it a promising candidate for integrated crop improvement strategies, aligning with the well-documented role of Streptomyces species in plant health promotion (Passari et al., 2019).

In addition to the strains with multifunctionality, our study also identified several bacterial strains with notable PGP potential for tomato cultivation. In particular, S. olivaceus (BHM1), S. variegatus (BHM3), B. stercoris (BHR2), and M. osloensis (YHT4-1) demonstrated significant positive effects on all three measured growth parameters: fresh weight, stem length, and root length (Fig. 5). These strains could be considered as prime candidates for growth-promoting inoculants specific to tomato plants.

The isolation and characterization of seed endophytic bacteria from Hawaii 7996 tomato seeds provided valuable insights into the antifungal potential of these endophytic bacteria against selected fungal pathogens. Paenibacillus was identified as the most dominant isolate, followed by Bacillus. The assessment of antifungal activity revealed that P. peoriae (YHR2-1) demonstrated inhibitory effects against F. oxysporum f. sp. lycopersici races 1 and 2, as well as B. cinerea (Fig. 6). This pattern of activity is consistent with other Paenibacillus species, which are known for their selective antifungal properties, particularly through the production of lipopeptides that disrupt fungal cell membranes (Kim et al., 2020). Such selective inhibition highlights the potential of Paenibacillus spp. as targeted biocontrol agents, offering a promising alternative to chemical fungicides in managing specific plant pathogens. Similarly, B. subtilis (BHN1) and B. stercoris (BHR2) exhibited significant antifungal activity against F. oxysporum f. sp. lycopersici races 1 and 2, as well as B. cinerea (Fig. 6). This finding aligns with previous research where Bacillus species, including B. subtilis and B. velezensis, have demonstrated strong antifungal properties against a range of plant pathogens, primarily through the production of antimicrobial compounds such as lipopeptides, which disrupt fungal cell membranes (Gao et al., 2017; Jamal et al., 2015; Khan et al., 2018). Several studies have investigated the potential of B. stercoris for biological control. For instance, Kim et al. (2015) reported that B. stercoris JS inhibited tobacco fungal and oomycete diseases by inducing plant resistance. Additionally, S. warneri (HSR_1) demonstrated antifungal activity against B. cinerea (Table 3), likely through the production of antimicrobial peptides. These peptides are recognized for their ability to inhibit a wide range of pathogenic microbes, positioning S. warneri as a promising biocontrol agent for managing fungal pathogens such as B. cinerea (Saha et al., 2022). Moreover, the inhibition of R. solani growth by B. subtilis has been attributed to the production of lipopeptides, such as iturin and surfactin, which are known to possess potent antifungal properties (Agarry et al., 2005; Melo, 1998; Montealegre et al., 2003; Yu et al., 2002).

This study demonstrates that tomato seeds harbor a diverse community of bacterial endophytes, which play crucial roles in seedling development, establishment, and protection against fungal pathogens. Our findings reveal that the seeds of the tomato cultivar Hawaii 7996 contain a diverse array of endophytic bacteria, several of which exhibit significant PGP properties. These endophytes may have a potential for enhancing crop resilience and productivity across various environmental conditions. The identified bacterial strains not only contribute to plant growth promotion but also provide a sustainable strategy to disease management in tomato cultivation. Future studies should aim to elucidate the mechanisms behind these beneficial interactions and developing practical applications for crop management.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

This work was supported by the Dong-A University research fund.

Fig. 1

Taxonomic distribution of endophytic bacteria isolated from Hawaii 7996 seeds. (A) Distribution of bacterial isolates at the phylum level. (B) Distribution of bacterial isolates at the family level.

Fig. 2

Indole-3-acetic acid (IAA) production by endophytic bacterial strains isolated from tomato seeds was detected using Salkowski’s reagent. The development of an orange-red color indicated the presence of IAA, highlighted by red boxes. The IAA-producing strains were identified based on the color change in the corresponding cuvettes. The inoculated bacterial strain is mentioned at the bottom of each cuvette, and the first plate on the left of each row is the control.

Fig. 3

Assessment of siderophore-producing ability of seed endophytic bacteria isolated from tomato cultivar Hawaii 7996 using the chrome azurol S agar assay. The formation of orange halos around bacterial colonies, highlighted by red boxes, indicates siderophore production by the endophytic strains. The inoculated bacterial strain is mentioned at the bottom of each plate.

Fig. 4

Assessment of phosphate-solubilizing ability of seed endophytic bacteria isolated from tomato cultivar Hawaii 7996 using modified Pikovskaya’s agar. The formation of clear halos around bacterial streaks indicates phosphate solubilization by the endophytic strains. The inoculated bacterial strain is mentioned at the bottom of each plate. Red boxes highlight the endophytic bacterial strains that exhibited phosphate solubilization.

Fig. 5

Evaluation of plant growth-promoting effects of bacterial strains on tomato cultivar Ye-gwang. The effects of bacterial treatments are shown for fresh weight (A), stem length (B), and root length (C) of Ye-gwang seedlings. Each box in the plot shows the median (central line), 25th percentile (lower edge of the box), and 75th percentile (upper edge of the box) of the fresh weight, stem length and root length data for each bacterial treatment. The whiskers extending from the boxes represent the range of the data, indicating the minimum and maximum values. Seedlings were treated with isolated bacterial strains and compared to an untreated control. Values represent the mean ± standard deviation of three replicates (n = 3). Statistically significant differences among treatments are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

Antifungal activity of seed endophytic bacteria isolated from tomato cultivar Hawaii 7996 against fungal pathogens Fusarium oxysporum f. sp. lycopersici race 1 (A), F. oxysporum f. sp. lycopersici race 2 (B), and Botrytis cinerea (C). The bacterial isolates exhibiting the strongest antifungal activity in the dual culture assay are shown. The inoculated bacterial strain is mentioned at the bottom of each plate.

Table 1

Identification of bacterial strains isolated from surface sterilized seeds of Hawaii 7996

Strain	Culture medium	Taxon name	Database	Similarity (%)	Hit strain
10HR1	1/10 R2A	Mycolicibacterium mucogenicum	NCBI	99.69	ATCC 49650
10HR2	1/10 R2A	Mycolicibacterium poriferae	NCBI	99.64	ATCC 35087
10HR3	1/10 R2A	Mycolicibacterium smegmatis	NCBI	99.78	NCTC 8159(T)
10HR4	1/10 R2A	Nocardioides marinus	NCBI	96.25	CL-DD14(T)
10HR6	1/10 R2A	Nocardioides aquiterrae	NCBI	98.9	GW-9(T)
10HT1	1/10 TSA	Peribacillus frigoritolerans	NCBI	100	DMS 8801
BHL1	LB	Brevibacillus limnophilus	NCBI	99.05	DSM 6472
BHM1	M9 salt	Streptomyces olivaceus	NCBI	99.93	NBRC 12805
BHM3	M9 salt	Streptomyces variegatus	NCBI	99.18	NBRC 15462
BHN1	NA	Bacillus subtilis	NCBI	99.93	168
BHN2	NA	Cohnella massiliensis	NCBI	99.57	6021052837
BHR2	R2A	Bacillus stercoris	NCBI	99.93	D7XPN1
BHT1	1/2 TSA	Fictibacillus enclensis	NCBI	98.99	NIO-1003
BHT2	1/2 TSA	Bacillus haynesii	NCBI	99.9	NRRL B-41327
HSR_1	R2A	Staphylococcus warneri	NCBI	99.58	ATCC 27836
HSR_2	R2A	Staphylococcus capitis subsp. capitis	NCBI	99.93	ATCC 27840
HSR_3	R2A	Cohnella suwonensis	NCBI	98.71	WD2-19
HSR_6	R2A	Micrococcus luteus	NCBI	99.64	NCTC 2665(T)
HSR_8	R2A	Bacillus acidicola	NCBI	98.64	105-2
HV17	V8A	Bacillus pseudomycoides	NCBI	99.79	NUQE_s
YHK1	King’s B	Priestia megaterium	NCBI	100	NBRC 15308
YHL1	LB	Metabacillus idriensis	NCBI	99.91	SMC 4352-2
YHL2	LB	Staphylococcus epidermidis	NCBI	99.92	NCTC 11047
YHN1-1	NA	Brevibacterium frigoritolerans	NCBI	100	DSM 8801
YHR1-1	R2A	Micrococcus yunnanensis	NCBI	99.43	YIM 65004
YHR2-1	1/2 TSA	Paenibacillus peoriae	NCBI	99.86	DSM 8320
YHT2-1	1/2 TSA	Bacillus infantis	NCBI	99.84	NRRL B-14911
YHT3-1	1/2 TSA	Paenibacillus barengoltzii	NCBI	99.93	NBRC 101215
YHT4-1	1/2 TSA	Moraxella osloensis	NCBI	99.62	CCUG 350
YHT5-1	1/2 TSA	Paenibacillus glucanolyticus	NCBI	99.86	DSM 5162
YHTT1	1/2 TSA	Paenibacillus cineris	NCBI	99.65	LMG 18439
YHTT2	1/2 TSA	Neobacillus drentensis	NCBI	99.37	LMG 21831
YHTT3	1/2 TSA	Neobacillus cucumis	NCBI	99.16	AP-6
YNS1-2	R2A	Priestia aryabhattai	NCBI	100	B8W22
YNS2-1	R2A	Paenibacillus provencensis	NCBI	100	4401170

R2A, Reasoner’s 2A agar; TSA, tryptic soy agar; LB, lysogeny agar; NA, nutrient agar; V8, V8 juice agar.

Table 2

Plant growth-promoting properties of bacterial endophytes isolated from surface sterilized seeds of Hawaii 7996

Strain	Taxon name	Production of indole 3 acetic acid (IAA)	Production of siderophores	Phosphate solubilizing	Plant growth promoting
10HR1	Mycolicibacterium mucogenicum	X^a	O	X	X
10HR2	Mycolicibacterium poriferae	X	X	X	X
10HR3	Mycolicibacterium smegmatis	X	O	O	X
10HR4	Nocardioides marinus	O^a	X	X	X
10HR6	Nocardioides aquiterrae	X	X	X	X
10HT1	Peribacillus frigoritolerans	O	X	X	O (FW^b, SL^b)
BHL1	Brevibacillus limnophilus	O	X	X	O (SL)
BHM1	Streptomyces olivaceus	O	X	X	O (FW, SL, RL^b)
BHM3	Streptomyces variegatus	O	O	O	O (FW, SL, RL)
BHN1	Bacillus subtilis	X	X	O	O (FW, SL, RL)
BHN2	Cohnella massiliensis	X	X	X	O (SL)
BHR2	Bacillus stercoris	X	X	O	O (FW, SL, RL)
BHT1	Fictibacillus enclensis	X	X	O	O (SL)
BHT2	Bacillus haynesii	X	X	O	X
HSR_1	Staphylococcus warneri	X	X	O	O (SL)
HSR_2	Staphylococcus capitis subsp. capitis	O	X	O	O (FW, RL)
HSR_3	Cohnella suwonensis	X	X	O	O (FW, RL)
HSR_6	Micrococcus luteus	O	X	X	O (FW, RL)
HSR_8	Bacillus acidicola	O	X	X	O (FW, RL)
HV17	Bacillus pseudomycoides	O	X	X	O (FW)
YHK1	Priestia megaterium	O	X	O	O (FW)
YHL1	Metabacillus idriensis	O	X	X	X
YHL2	Staphylococcus epidermidis	O	X	O	X
YHN1-1	Brevibacterium frigoritolerans	O	X	X	O (RL)
YHR1-1	Micrococcus yunnanensis	O	X	X	O (RL)
YHR2-1	Paenibacillus peoriae	X	O	O	X
YHT2-1	Bacillus infantis	X	X	X	X
YHT3-1	Paenibacillus barengoltzii	X	X	X	O (FW, RL)
YHT4-1	Moraxella osloensis	O	X	X	O (FW, SL, RL)
YHT5-1	Paenibacillus glucanolyticus	O	X	X	O (FW, RL)
YHTT1	Paenibacillus cineris	O	X	X	X
YHTT2	Neobacillus drentensis	X	X	X	X
YHTT3	Neobacillus cucumis	O	X	X	X
YNS1-2	Priestia aryabhattai	O	X	X	X
YNS2-1	Paenibacillus provencensis	X	X	X	O (FW, SL)

^a O, presence of biological activity (e.g., indole-3-acetic acid [IAA] production, siderophore production, phosphate solubilizing, or plant growth-promoting effect); X, absence of biological activity (e.g., IAA production, siderophore production, phosphate solubilizing, or plant growth-promoting effect).

^b FW, significantly increased the fresh weight of tomato plants compared to the control (sterile distilled water); SL, significantly increased the stem length of tomato plants compared to the control (sterile distilled water); RL, significantly increased the root length of tomato plants compared to the control (sterile distilled water).

Table 3

Antifungal activities of bacterial endophytes isolated from surface sterilized seeds of Hawaii 7996

Strain	Taxon name	Fusarium oxysporum race 1	Fusarium oxysporum race 2	Rhizoctonia solani	Botrytis cinerea
10HR1	Mycolicibacterium mucogenicum	−^a	−	−	−
10HR2	Mycolicibacterium poriferae	−	−	−	−
10HR3	Mycolicibacterium smegmatis	−	−	−	−
10HR4	Nocardioides marinus	−	−	−	−
10HR6	Nocardioides aquiterrae	−	−	−	−
10HT1	Peribacillus frigoritolerans	−	−	−	−
BHL1	Brevibacillus limnophilus	−	−	−	−
BHM1	Streptomyces olivaceus	−	−	−	−
BHM3	Streptomyces variegatus	−	−	−	+
BHN1	Bacillus subtilis	++ ^a	++	+	++
BHN2	Cohnella massiliensis	−	−	−	−
BHR2	Bacillus stercoris	++	++	+	++
BHT1	Fictibacillus enclensis	−	−	−	−
BHT2	Bacillus haynesii	−	−	−	−
HSR_1	Staphylococcus warneri	−	−	−	+
HSR_2	Staphylococcus capitis subsp. capitis	−	−	−	−
HSR_3	Cohnella suwonensis	−	−	−	−
HSR_6	Micrococcus luteus	−	−	−	−
HSR_8	Bacillus acidicola	−	−	−	−
HV17	Bacillus pseudomycoides	−	−	−	−
YHK1	Priestia megaterium	−	−	−	+
YHL1	Metabacillus idriensis	+ ^a	−	−	−
YHL2	Staphylococcus epidermidis	−	−	−	−
YHN1-1	Brevibacterium frigoritolerans	−	−	−	−
YHR1-1	Micrococcus yunnanensis	−	−	−	−
YHR2-1	Paenibacillus peoriae	++	++	−	++
YHT2-1	Bacillus infantis	−	−	−	−
YHT3-1	Paenibacillus barengoltzii	−	+	−	−
YHT4-1	Moraxella osloensis	−	+	−	−
YHT5-1	Paenibacillus glucanolyticus	+	−	−	−
YHTT1	Paenibacillus cineris	−	−	−	−
YHTT2	Neobacillus drentensis	−	−	−	−
YHTT3	Neobacillus cucumis	+	−	−	−
YNS1-2	Priestia aryabhattai	−	−	−	+
YNS2-1	Paenibacillus provencensis	−	−	−	−

^a ++, strong antifungal activity; +, weak antifungal activity; −, no activity.

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