Isolation and Characterization of Plant Growth-Promoting Rhizobacteria from Mud Flats in South Korea: Potential for Lettuce Growth Promotion and Control of Sclerotinia Rot

Abstract

Various strategies have been developed to control lettuce diseases on farms and in food-packing plants. Biological control is considered a promising alternative owing to its eco-friendly nature. In the present study, bacteria isolated from coastal mudflats were evaluated for their efficacy in controlling Sclerotinia rot, and the plant growth-promoting activity in lettuce was also assessed. Among the screened microorganisms from the coastal mudflats, 12 bacterial strains exhibited antifungal activity against Sclerotinia sclerotiorum selected. These isolates have shown beneficial characteristics, such as nitrogen fixation, indole-3-acetic acid production, phosphate solubilization, and siderophore production. Additionally, the selected isolates showed antifungal effects on the pathogens of major plant disease, such as Alternaria porri, Colletotrichum acutatum, Fusarium oxysporum, Phytophthora capsici, Pythium ultimum, Rhizoctonia solani, and Stemphylium lycopersici. Among the selected bacterial strains, Bacillus subtilis GCM190 exhibited a high sclerotinia rot control rate, similar to that of the tebuconazole-treated group, and showed a significant effect in promoting the growth of lettuce leaves, stems, and roots (least significant difference, P = 0.05). The selection of rifampicin-resistant mutants and their tracing on lettuce roots and soil confirmed that they were well established in both the soil and lettuce roots. The selected microorganisms also exhibited antifungal effects in vitro against other crop diseases affecting cucumbers, tomatoes, red peppers, and green onions, suggesting high potential for practical applications.

Lettuce, Lactuca sativa L. (Asteraceae), is one of the most widely consumed leafy vegetables worldwide (Petkova and Dimova, 2024). Among the various lettuce diseases, lettuce drop caused by Sclerotinia spp. is especially destructive in major lettuce-growing regions (Hao et al., 2003). Sclerotinia sclerotiorum, the causative agent of basal drop and white mold, causes necrotic lesions, white mycelial mats, and black sclerotia in lettuce (Shrestha et al. 2018). This pathogen spreads rapidly in plants and causes severe economic losses (Aguilar-Pérez et al., 2023; Purdy 1979; Rakesh et al., 2016; Terrones-Salgado et al., 2023). Although fungicides have long been used to prevent plant diseases and improve productivity (Gulzar et al., 2023), exposure to these chemicals poses serious health risks including nausea, dizziness, skin irritation, developmental disorders, and neurodevelopmental defects. Long-term exposure may lead to genetic mutations and immune system impairment (AbuQamar et al., 2024; Brenet et al., 2020; Frazier, 2007; Sinambela, 2024). Additionally, pesticide contamination contributes to ecosystem degradation and pollution, which has prompted regulatory measures and a reduction in the use of highly toxic fungicides in developed countries (Frazier, 2007). These environmental and health concerns have driven the search for sustainable alternatives (Gulzar et al., 2023).

Organic farming is a key alternative that reduces pollution, conserves water, and enhances soil fertility (Yadav, 2023). Organic foods contain fewer harmful residues (Saikanth et al., 2023), which lowers the risk of obesity, certain cancers, and nutrient deficiencies (Rahman et al., 2024). In addition, such foods are rich in vitamins and antioxidants and are increasingly preferred, especially in high-income and health-conscious populations (Mingazova et al. 2024; Rakhaeva and Mizyureva, 2024). The organic food market, which is now a multi-billion dollar industry (Mingazova et al., 2024), benefits small-scale farmers by enabling premium pricing and economic sustainability (Varma et al., 2024). With the commencement of the U.S. Department of Agriculture school meal standards program, salad consumption in elementary schools in the United States increased from 17.1% (2006–2007) to 29.6% (2012–2013) (Ohri-Vachaspati et al., 2016). However, as salad consumption continues to increase in modern society, foodborne illnesses associated with leafy vegetables such as lettuce have also increased (Cufaoglu et al., 2017), highlighting the growing importance of growing healthy lettuce without the use of fungicides.

Antagonistic microorganisms are important in agriculture because they control plant disease pathogens and promote plant growth through phosphate and sulfate solubilization, production of growth regulators, such as indole-3 acetic acid (IAA), siderophore production, nitrogen fixation, denitrification, immune modulation, and disease control (Ji et al., 2014; Sharma et al., 2022). Biological control agents derived from strains of Bacillus, Pseudomonas, and Streptomyces are eco-friendly alternatives to chemical fungicides that significantly inhibit plant pathogens (Ahmed et al., 2024). Among these, soil-derived Bacillus strains have shown better antifungal effects than amphotericin B and have high potential as biocontrol agents (Elamin et al. 2021). Additionally, the antifungal agents produced by Bacillus strains have been found to be noncytotoxic, indicating their potential for safe use (Ye et al., 2023). Microorganisms play crucial roles in nutrient mobilization, absorption, growth promotion, disease suppression, and nutrient enrichment in plants (Sharma et al. 2022). IAA enhances plant height, root and stem thickness, and chlorophyll content, and improves yield and nutrient accumulation (Li et al., 2024; Liu et al., 2023). Additionally, the expression of genes related to IAA biosynthesis and transport increases, thereby enhancing stress tolerance mechanisms (Ma et al., 2022). Nitrogen is essential for the synthesis of proteins, nucleic acids, and chlorophyll, all of which are important for plant growth. It also regulates plant hormones and microRNAs, thereby influencing root development and stress responses (Wang et al. 2024). Through these functions, nitrogen significantly affects yield parameters such as grain weight (Markad, 2024; Yadav, 2024). Siderophores are essential for plant metabolism and serve as cofactors in various physiological and biochemical processes including photosynthesis and respiration (Bhat et al., 2024). An inadequate level of siderophores reduces photosynthetic rates and inhibits chlorophyll biosynthesis, leading to chlorosis, a condition characterized by leaf yellowing (Saha and Singh, 2024; Tabata, 2023). Phosphorus affects the development and functioning of stomata, regulates plant physiological responses to abiotic stresses, such as drought and salinity (Khan et al., 2023). However, plant uptake of phosphorus is limited by environmental and soil factors, such as temperature, water availability, and soil acidity (Grzebisz et al., 2024). Iron and phosphorus in the soil are often present in insoluble forms, which inhibits plant uptake of these nutrients (Bhat et al., 2024; Négrel et al., 2024).

More than 80% of the bacterial strains isolated from mudflats, at a depth of 5 to 25 cm, were identified as Bacillus spp. (Noh, 2016), as is also the case in hypersaline environments such as the Dead Sea (Jacob 2012). These findings suggest that mudflat microbes possess enhanced stress tolerance owing to their unique environmental conditions (Zhang et al., 2023).

In the present study, bacteria were isolated from coastal mudflats, which are considered a more challenging environment than terrestrial soil, to identify antagonistic microorganisms with greater soil adaptability. Coastal mudflats are characterized by high salinity, low oxygen levels, and dynamic nutrient availability, conditions that foster microbial communities with enhanced stress tolerance and metabolic versatility. These extreme environments are therefore promising reservoirs for the discovery of robust biocontrol agents. Despite their potential, mudflat-derived antagonistic bacteria have been largely underexplored compared to those isolated from conventional agricultural soils. In this study, the potential of the isolates as antagonistic microorganisms for reducing chemical pesticide use and supporting sustainable agriculture was evaluated through their efficacy in controlling Sclerotinia rot and promoting lettuce growth.

Materials and Methods

Isolation of antagonistic and endophytic microorganisms

Soil samples were collected from sea mudflats and rhizospheres of lettuce and mixed with 45 mL of distilled water per 5 g. Root samples were surface-sterilized with 1% sodium hypochlorite for 1 min to remove epiphytic microorganisms. Both soil and root samples were then serially diluted from 10⁻¹ to 10⁻⁵. Subsequently, 100 μL of each dilution was spread onto potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) and tryptic soy agar (TSA; Difco Laboratories) plates. The plates were incubated at 25°C for 2 days to obtain pure isolates. Endophytic bacteria were isolated from lettuce roots as described by Ji et al. (2014).

Replacement culture with Sclerotinia sclerotiorum was performed to select bacteria that were determined to have antifungal activities, and the antifungal effects of the selected bacteria were tested on various crop fungal diseases. The plant pathogens used in the experiment were obtained from the National Institute of Agricultural Sciences of the Rural Development Administration, South Korea.

Antifungal effect of selected bacteria in vitro on plant pathogens

The antifungal activity of bacteria isolated from various soils was measured on major plant disease pathogens such as Alternaria porri, Colletotrichum acutatum, Fusarium oxysporum, Phytophthora capsici, Pythium ultimum, Rhizoctonia solani, and Stemphylium lycopersici. A mycelium agar plug (5 in diameter) was placed at the center of the agar plate by dispensing PDA in a 90 mm diameter Petri dish (SPL Life Sciences, Seoul, Korea). The selected antagonistic microorganisms were inoculated 10 mm from the end of the Petri dish. As a control, only agar blocks of pathogens were inoculated. After the control was sufficiently grown in Petri dishes, the distance between the antagonistic microorganisms and the mycelium was measured to check the antifungal effect. All plant pathogens used in the experiments were obtained from the Agricultural Type Collection Center of the National Institute of Agricultural Sciences of the Rural Development Administration, South Korea.

Characterization of antagonistic microorganisms as plant growth promoting rhizobacteria

Auxin activity test

To measure the production of the plant hormone IAA, a 0.1% tryptophan solution was added to King’s B medium (2% proteose peptone, 0.25% K₂HPO₄, 0.6% MgSO₄, 1.5% glycerol, pH 7.2), and separated strains were inoculated and cultured at 30°C for 2 days. After centrifugation (10,000 rpm, 4°C, 15 min), the supernatant was separated, and Salkowski’s reagent (35% HClO₄ 50 mL, 0.5 M FeCl₃ 1 mL) was mixed in a 1:2 (v/v) ratio, followed by incubation at 25°C for 30 min until a pink color appeared. The degree of coloration was measured at 530 nm using a Multiskan GO spectrophotometer (Multiskan GO, Thermo Scientific, Vantaa, Finland). Using IAA as the standard substance, a calibration curve was determined following the same procedure as described previously, and the concentrations of the sample were calculated (Leveau and Lindow, 2005).

Phosphate-solubilizing activity

The phosphate-solubilizing activities of the strains demonstrating antifungal effects were determined using the methods described by Mehta and Nautiyal (2001) and Rodríguez and Fraga (1999). National Botanical Research Institute’s phosphate growth (NBRIP) agar plates (10 g glucose, 5 g Ca₃(PO₄)₂, 5 g MgCl₂·6H₂O, 0.25 g MgSO₄·7H₂O, 0.2 g KCl, and 0.1 g (NH₄)₂SO₄, 1.5 g water agar [per liter], pH 7) insoluble tricalcium phosphate (Ca₃(PO₄)₂) as the sole phosphorus source were used to detect phosphate solubilizing activity. Approximately 40 μL of bacterial suspension grown for 48 h was spread on NBRIP agar plates using a spreader and incubated at 30°C for at least 1 week. The appearance of clear zones around the bacterial colonies indicated phosphate-solubilizing activity owing to the production of organic acids by the bacteria.

In addition to qualitative screening, quantitative experiments were conducted to assess and select the microorganisms that produced halos or clear zones. Bromophenol blue (NBRI-BPB) broth, composed of NBRIP medium supplemented with 0.025 g/L bromophenol blue, was used for quantitative assays. Bacterial isolates were pre-cultured for 2 days in tryptic soy broth (TSB; Difco Laboratories) before being transferred into NBRI-BPB broth. The cultures were incubated at 30°C for 3 days with shaking at 250 rpm. After incubation, the cultures were harvested by centrifugation at 5,000 rpm for 20 min, and the absorbance of the supernatant was measured at 600 nm to determine phosphate-solubilizing activity. P. aeruginosa (KACC 11085) was used as positive control in these experiments.

Isolation of endophytic bacteria and their activity tests

The nitrogen fixation ability of the selected bacteria was determined according to the method described by Ji et al. (2014). The strains used in the experiment were inoculated into TSB and cultured at 30°C with shaking at 180 rpm for 1 day. After centrifugation, the bacterial cells were collected using a loop and washed twice with nitrogen-free bromothymol blue (NFB; 0.5% HO₂CCH₂CH(OH)CO₂H, 0.05% K₂HPO₄, 0.001% MgSO₄·7H₂O, 0.002% NaCl, 0.005% FeSO₄·7H₂O, 0.0002% Na₂MoO₄, 0.001% MnSO₄·7H₂O, 0.001% CaCl₂, 0.4% KOH, 0.2% bromothymol blue [in 0.5% alcohol], 0.175% agar, pH 6.8), and then inoculated into 7 mL of NFB agar. Bacillus subtilis CB-R05 from Ji et al. (2014) was used as a positive control. The fully grown strain was inoculated into NFB agar in test tubes and incubated at 30°C for 4 days. A positive result was determined when the color of the medium turned blue.

Siderophore-producing activity

Siderophore-producing bacteria were selected from antagonistic microorganisms that target turfgrass diseases, using the method described by Schwyn and Neilands (1987). This assay identifies siderophore producers based on a color change from blue to another color in the medium. The experiment was conducted according to the methods described by Milagres et al. (1999) and Ji et al. (2014) with slight modifications. Three types of solutions were used to prepare 1 L of Chrome Azurol S (CAS) blue broth. To prepare solution A, which was the Fe-CAS indicator solution, 60.5 mg) was dissolved in 50 mL of water and mixed with 10 mL of an iron (III) solution (1 mM FeCl₃·6H₂O, 10 mM HCl). With stirring, this solution was slowly added to 72.9 mg of HDTMA (hexadecyltrimethylammonium bromide) dissolved in 40 mL of water. The resulting dark blue liquid was autoclaved and cooled to 55°C. For buffer solution (2), 30.24 g of PIPES was dissolved in 900 mL of salt solution containing 0.3 g KH₂PO₄, 0.5 g NaCl, and 1.0 g NH₄Cl, and the pH was adjusted to 6.8 using KOH solution. Subsequently, King’s B medium (2% proteose peptone, 0.25% K₂HPO₄, 0.6% MgSO₄, and 1.5% glycerol per 1 L) was added. After adding 1.5 g of agar, solution B was autoclaved and cooled to 55°C. Once both solutions A and B were autoclaved and cooled to 55°C, they were transferred to a light-shielding glass bottle, mixed thoroughly, and used as CAS blue broth. Subsequently, 4 mL of the prepared broth was dispensed into conical centrifuge tubes and one loop of antagonistic microorganisms was inoculated. The inoculated tubes were incubated at 30°C with shaking at 250 rpm for 3 days, and the breakdown or disappearance of the blue color was observed to confirm siderophore production by the bacteria.

Rhizosphere colonization ability of antagonistic microorganisms

Antagonistic microorganisms were spread on PDA plates supplemented with 50 ppm rifampicin and spontaneous colonies were selected. Subsequently, the harvested colonies were alternately streaked three times on PDA and PDA supplemented with 50 ppm rifampicin to confirm that the selected antagonistic microorganisms had acquired rifampicin-resistant mutations. The selected rifampicin-resistant mutant strains were cultured on TSA plates for 2 days and harvested using a spreader, and the culture concentration was adjusted to an optical density (O.D.) of 0.1 using distilled water. The O.D. density was measured using a spectrophotometer (Multiskan GO, Thermo Scientific), and 30 mL of the 0.1 O.D. rifampicin-resistant mutant strain suspension was applied to each pot containing lettuce plants. Colonization of the mutant strains in the rhizosphere was observed immediately after application until 6 d post-treatment. To evaluate rhizosphere colonization, 5 g of root rhizosphere soil and roots were collected from each pot and serially diluted to 10 U using distilled water. The diluted samples were spread on PDA plates supplemented with 50 ppm rifampicin, and colony-forming units (CFU) were counted to assess the colonization ability of the mutant strains.

Microbial identification

16S rRNA gene analysis was performed to identify the isolated strains. The 16S rRNA gene was amplified using the universal primers 785F (GGATTAGATAACCCTGGTA) and 907R (CCGTCAATTCMTTTTRAGTTT), and the analysis was performed by Macrogen (Seoul, Korea). Sequence analysis was carried out using the CLC workbench software v.8.0 and aligned with representative members of the selected genera using the CLUSTAL W program. A phylogenetic tree was constructed to identify the datasets using the neighbor-joining method with CLC Workbench software v.8.0.

Pot experiments: effect of selected microorganisms on Sclerotinia rot and lettuce growth

The pot experiment was conducted as a pilot study in a hydroponic growth chamber in a plant growth room at Konkuk University from November 1, 2023, to November 29, 2023 (Fig. 1). Subsequently, the main experiment was conducted in a greenhouse at Konkuk University from February 24, 2024, to April 20, 2024. Potting soil (Tobaeki, (C)Sunghwa, Boseong, Korea) was sterilized by autoclaving twice at 121°C for 40 min and then transferred to pots (diameter 70 mm, height 100 mm). Lettuce seeds (cultivar Cheong Chi Ma) were sown in pots in a greenhouse, and the plants were used for the experiments when they reached the six-leaf stage after germination. The microorganisms used in this study were the unidentified species of the strain GCM082 and Bacillus subtilis GCM190. To treat the microorganisms, each isolate was cultured on TSA for 24 h and harvested using a spreader, and their O.D. was adjusted to 0.1 O.D. at 600 nm using a spectrophotometer and 3rd distilled water. Subsequently, 30 mL of the culture was treated into each pot, and the experiments were conducted. A 20% fungicide (Silbaco Plus, a. i., tebuconazole, Bayer Korea, Seoul, Korea) was used as a control, and the mortality rate of lettuce was determined to calculate the disease control rate for each treatment group.

Fig. 1.

Pilot experiment on the effects of selected antagonistic microorganisms on lettuce growth and control of Sclerotinia rot in the hydroponic growth chamber. Before treatment (A), 10 days after the first treatment (B).

For inoculum preparation, rice seeds were sterilized by autoclaving twice at 121°C for 40 min. S. sclerotiorum mycelia were inoculated onto PDA and allowed to grow. Fresh mycelial plugs of S. sclerotiorum grown on agar were transferred to sterilized rice seeds to prepare the inoculum. The inoculated rice seeds were incubated at room temperature for the full growth of S. sclerotiorum mycelia. After incubation, the prepared inoculum was dried to prevent bacterial contamination and stored for experimental use. For pathogen inoculation in the lettuce pots, two rice seeds inoculated with Sclerotinia rot were inserted at two points, each located 10 mm away from the lettuce stem and at a depth of up to 10 mm.

All treatments were performed in triplicates. Chlorophyll content was measured using a SPAD-502Plus (Konica Minolta, Tokyo, Japan). The experiment for testing the effect of plant growth promoting rhizobacteria (PGPR) was conducted by measuring the growth of leaves and chlorophyll content of lettuce in the pots every week after microbial treatment. Two weeks after the last microbial treatment, the dry weights of the roots, stems, and leaves were measured. In all experiments, each pot received 30 mL of water every 2 days. The pilot experiment was conducted with four replicates of pots per treatment, whereas the main greenhouse experiment was conducted with 10 pots per treatment in four replicates (total of 40 pots) with a randomized block design. The disease control rate was measured based on the time point at which almost half of the lettuces in the pathogen-inoculated treatment group showed mortality.

Results and Discussion

Isolation of antagonistic microorganisms

All bacterial isolates were screened through dual culture assays against S. sclerotiorum to identify those that exhibited antifungal effects. As a result, 12 antagonistic microorganisms were selected.

Antifungal effect of selected bacteria in vitro on the plant pathogens

Dual-culture assays are widely employed as an in vitro method for the initial screening of biological control agents (Desai et al., 2002; Ji et al., 2014). Among the 12 bacterial isolates from various sources that exhibited antifungal effects against S. sclerotiorum, the top five bacteria with the widest inhibition zones were selected (Fig. 2). These selected bacteria were tested for in vitro antifungal effects against the major disease-causing pathogens in plants, namely A. porri, C. acutatum, Fusarium oxysporum, Phytophthora capsici, Pythium ultimum, R. solani, S. sclerotiorum, and Stemphylium lycopersici (Table 1). All five bacterial isolates exhibited antagonistic effects by inhibiting the mycelial growth of all plant pathogens tested.

Fig. 2.

Experiment for selecting antagonistic microorganisms with both antifungal and PGPR effects. Greater distance between the fungus and bacteria was considered indicative of high antifungal activity, and this was used as a basis for selecting candidate antagonistic microorganisms with antifungal effects (A). Halo zone was considered indicative of phosphate-solubilizing activity, and a larger halo zone was considered indicative of higher phosphate-solubilizing activity (B). Halo zone was considered indicative of siderophore production, and a larger halo zone was considered indicative of higher siderophore production (C). The broth inoculated with antagonistic microorganisms turning from yellow to blue was considered indicative of nitrogen-fixing ability, and deeper blue color was considered indicative of higher nitrogen-fixing activity (D).

Table 1.

In vitro antifungal effects of bacterial isolates from mud flat against plant pathogens

Strain	In vitro antifungal effect of the bacterial isolates on plant pathogen represented in the zone of inhibition (size in mm)
	Lettuce (Lactuca sativa)				Red pepper (Capsicum annuum L.)		Welsh onion (Allium fistulosum L.)		Tomato (Solanum lycopersicum L.)				Cucumber (Cucumis sativus L.)
	Sclerotinia sclerotiorum	R. solania AG-1(IB)	F. oxysporum	Phytophthora sp.	P. capsici	C. acutatum	R. solani AG-4	A. porri	P. capsici	C. acutatum	S. lycopersici	C. coccodes	R. solani AG-1(IB)	P. capsici	Pythium ultimum	F. oxysporum
GCM076	5.6 bb	1.3 b	5.7 b	2.7 c	7.4 b	7.5 c	3.3 b	2.9 b	7.2 b	7.1 c	7.6 b	9.6 b	5.7 a	7.3 a	1.1 a	4.6 b
GCM082	10.3 a	1.4 b	7.4 a	5.3 b	3.7 d	7.5 c	3.51 b	1.0 d	7.2 b	4.5 d	6.1 bc	8.0 c	5.7 a	7.2 a	1.1 a	4.9 bc
GCM136	0.5 c	-c	3.0 c	-	4.7 c	11.0 a	4.7 a	2.6 bc	8.4 a	8.2 a	9.8 a	10.6 a	6.0 a	7.2 a	1.0 a	7.5 a
GCM190	10.5 a	2.2 a	3.0 c	8.8 a	8.6 a	8.7 b	-	4.6 a	8.1 a	7.5 b	9.0 ab	10.5 a	-	4.6 b	1.1 a	7.8 a
GCM219	0.5 c	-	2.9 c	-	4.7 c	5.9 d	-	2.3 c	-	4.0 e	3.3 c	7.8 c	-	-	1.0 a	5.5 c

^aR, Rhizoctonia; F, Fusarium; C, Colletotricum; A, Alternaria; and S, Septoria.

^bMeans followed by different letters in each column indicate significant difference by Fisher’s least significant difference (P = 0.05).

^cNo antifungal effect.

Characterization of antagonistic microorganisms as PGPR

Production of auxin

IAA is an important auxin in plants that regulates various critical physiological functions such as cell growth and division, tissue specialization, and phototropic responses (Frey-Klett et al., 2005; Gordon and Weber, 1950; Ji et al., 2014; Khalid et al., 2004; Leveau and Lindow, 2005). Of the 12 isolates, 11 produced high auxin levels. These isolates produced different amounts of auxin, ranging from 9.3 to 19.3 μg/mL (Table 2). In contrast, only one isolate, ICBR02 showed relatively low auxin production, producing less than 4.6 μg/mL. Notably, GCM190 was identified as a highly efficient IAA-producing strain, exhibiting higher IAA production than the values previously reported by Ji et al. (2014), Khalid et al. (2004), Ng et al. (2012), and Yasmin et al. (2007). We conducted a detailed investigation to evaluate whether the application of the selected antagonistic microorganisms could enhance the growth of other crops, such as lettuce, by increasing IAA production. However, Salkowski’s method may not be suitable for quantifying IAA production. The Salkowski reagent was suitable for detecting IAA within the range of 0.5–200 μg/mL, but it also reacted with indolepyruvic acid and indoleacetamide, not just IAA (Glickmann and Dessaux, 1995). The Salkowski reagent specifically reacts with indolepyruvic acid and indoleacetamide, in addition to IAA, indicating that this method can also reflect the presence of other indole compounds (Glickmann and Dessaux, 1995). A comparative analysis of IAA production using the Salkowski method and LC-MS/MS (liquid chromatography–tandem mass spectrometry) revealed that the Salkowski method may produce higher IAA concentrations than the actual levels because of interactions with other indole compounds (Gang et al., 2019). It has been reported that the Salkowski reagent overestimates IAA concentrations by 41–1,042 times in the absence of tryptophan, and by 7–330 times when tryptophan is present as an inducer (Guardado-Fierros et al., 2024). These studies suggest that the Salkowski method may have limitations in accurately quantifying IAA. The Salkowski method is considered suitable for the qualitative screening of IAA-producing strains. Future studies will require more appropriate and sensitive techniques and methodologies to quantify IAA and other auxins.

Table 2.

Different biological activities of the bacterial isolates from diverse sources on the plant growth or antifungal related

Strain	Biological activity
	IAA (μg/mL)a	Solubilized phosphate (μg/mL)b	Nitrogen fixationc	Siderophore productiond
BSG011	10.3	0.6	−e	+
GCM076	18.5	0.3	−	−
GCM082	13.5	1.2	−	+
GCM136	10.2	0.6	−	−
GCM190	19.3	1.4	−	+
GCM219	11.3	0.3	−	−
HRP003	10.1	1.2	−	+
ICBR02	4.6	0.4	+	+
IKBR40	9.3	0.3	+	+
IMS042	10.0	0.7	−	+
IMS064	9.6	0.4	−	+
IZGR24	12.5	0.3	+	−

^aIndole-3 acetic acid (IAA) was determined at more than 16 h after the bacterial growth in King’s B supplemented with 0.1% tryptophan with absorbance at 530 nm.

^bPhosphate-solubilizing activities of the isolates were tested by inoculating each bacterial strain on National Botanical Research Institute’s phosphate growth (NBRIP) agar plate and determined with absorbance at 600 nm.

^cBacteria cultured for 1 day were inoculated on the nitrogen-free bromothymol blue broth and the color change of the broth was observed for 1 week; +, positive; −, negative.

^dThe clear zone was confirmed by inoculating 100 μL of bacterial suspension onto Chrome Azurol S blue agar plates followed by incubating at 30°C for 48 h.

^e+, positive; −, negative.

Phosphate-solubilizing activity

The release of insoluble forms of phosphorus plays a crucial role in enhancing the availability of phosphorus in the soil (Rodriguez and Fraga, 1999). The application of phosphate-solubilizing bacteria as bioinoculants can promote both phosphorus uptake by plants and crop productivity (Mehta and Nautiyal, 2001). Several bacterial genera, including Bacillus, Pseudomonas, Serratia, and Enterobacter, can dissolve insoluble phosphate compounds, thereby supporting plant growth (Frey-Klett et al., 2005; Hameeda et al., 2008). Of the 12 isolates tested, four demonstrated phosphate-solubilizing ability by forming clear halos on NBRIP agar plates (Fig. 2). These four isolates solubilized phosphate in varying amounts, ranging from 0.7 to 1.4 μg/mL (Table 2). This indicates that antagonistic microorganisms exhibiting antifungal activity against fungal plant pathogens can produce beneficial properties that promote plant growth.

Nitrogen fixing activity

The nitrogen fixation ability was determined according to the method described by Um et al. (2014). Among the 12 isolates used in the experiment, three isolates (Serratia liquefaciens ICBR02, Ewingella americana IKBR40, and Serratia liquefaciens IZGR24) exhibited nitrogen-fixing ability, resulting in yellow NFB medium to blue, as shown in Table 2 and Fig. 2. These were endophytic bacteria isolated from the roots of creeping bentgrass, Kentucky bluegrass, and zoysia grass. Nitrogen is an important element in plants because it is a component of proteins, nucleic acids, and various biomolecules (Seefeldt et al., 2009). The main forms of nitrogen absorbed by plant roots are inorganic nitrogen, known as nitrate nitrogen (NO₃⁻) and ammonium nitrogen (NH₄+) (Li et al., 2013). Ji et al. (2014) reported that an endophytic strain isolated from rice roots exhibited both nitrogen-fixing and antifungal effects. Turfgrass belongs to the same family as rice (Oryza sativa), suggesting that nitrogen-fixing endophytic bacteria may also be present in turfgrass roots.

Siderophore production

Among the 12 selected bacterial isolates, eight (Bacillus amyloliquefaciens BSG011, Bacillus amyloliquefaciens IMS042, Bacillus subtilis GCM190, Bacillus cereus HRP003, Serratia liquefaciens ICBR02, Ewingella americana IKBR40, Pseudomonas iranensis IMS064, and the unidentified species of the strain GCM082) were confirmed to produce siderophores through the CAS-blue agar assay (Table 2). These isolates exhibited a color change from blue to orange (Fig. 2), indicating siderophore production. B. subtilis CB-R05, which was used as comparative control, was similarly confirmed to produce siderophores. Siderophores play an important role in plant metabolism and growth, as they regulate reactive oxygen species, electron transport, metabolic processes, and redox reactions (Li et al., 2013). Owing to the limited availability of iron for microbial and plant growth in the soil, rhizosphere microorganisms residing around plant roots produce siderophores with low molecular weights (400–1,000 Da) to enhance iron acquisition by microbes and plants (Hider and Kong, 2010). Previous studies have reported that siderophore-producing bacteria significantly improve the absorption of various metals such as Fe, Zn, and Cu in plants (Carrillo-Castañeda et al., 2005; Dimkpa et al., 2008, 2009; Egamberdiyeva, 2007; Gururani et al., 2012). Siderophores not only increase bacterial access to essential minerals, but also directly stimulate the biosynthesis of other antimicrobial compounds, thereby inhibiting pathogenic fungi such as F. oxysporum and R. solani. These siderophores act as stress factors that trigger host resistance mechanisms (Haas and Défago, 2005; Joseph et al., 2007; Wahyudi et al., 2011). The typical color change associated with siderophore production described in the literature results from the sequestration of iron from CAS by siderophores (Schwyn and Neilands, 1987). However, the rate of color change in solid media does not provide an exact quantification of siderophore production (Ji et al., 2014). Siderophores are produced by plant growth-promoting bacteria (PGPB), such as Pseudomonas spp., which colonize plant root surfaces, facilitate iron absorption, and inhibit pathogenic microorganisms (Seong and Shin, 1996). PGPB isolates with high siderophore-producing activity should be further studied for their potential to confer disease resistance in higher plants.

Soil colonization ability of selected antagonistic microorganisms

Among the strains isolated from various sources, GCM072, GCM082, GCM136, GCM190, and GCM219 were isolated from mudflats in Gochang, South Korea. GCM076, GCM082, and GCM190 exhibited the highest antifungal activities. In addition, these isolates were well-established in the soil and maintained their populations. In addition, the antagonistic microorganisms were confirmed to have been successfully established in the rhizosphere of lettuce, as shown in Table 3 and Fig. 3. Soil salinity dissolved organic carbon, and elevated CO₂ concentrations significantly influence the diversity and composition of microbial communities in wetlands such as mud flats, indicating that tidal flat microorganisms can adapt to these changing environmental conditions in the long term (Yang et al., 2023; Zhang et al., 2023). The soil adaptability of antagonistic microorganisms is essential for their coevolution with native soil microbes and enhances disease suppression through complex microbial interactions (Kinkel et al., 2011). Bacillus and Pseudomonas strains have demonstrated significant adaptability and efficacy in controlling potato pathogens, indicating their potential as biological control agents in various soil types (Caulier et al., 2018).

Table 3.

Colonization of different rifampicin-resistant mutant antagonists on the rhizosphere of lettuce in pot experiment

Treatment	Population of rhizosphere microorganism (CFU/g) × 106
	0 day	1 day	2 days	4 days	6 days
GCM076	4.0 aa	41.0 a	74.7 a	28.0 b	35.0 a
GCM082	4.0 a	46.7 a	17.7 b	48.0 a	37.7 a
GCM190	6.7 b	5.7 b	12.0 b	5.3 c	9.3 b

^aMeans followed by the same letters are not significantly different within the column at P = 0.05 (least significant difference).

Fig. 3.

The selected rifampicin-resistant mutant antagonistic microorganisms (CFU/g) × 10⁶ were inoculated onto potato dextrose agar supplemented with 50 ppm rifampicin and incubated at 25°C for 2 days. GCM190 (A), GCM082 (B), GCM076 (C), and untreated (D).

Identification of selected antagonistic microorganisms using 16S rDNA

Through 16S rDNA sequence analysis, 12 selected antagonistic bacterial candidates were identified as belonging to two species of Serratia, one species of Ewingella, one species of Pseudomonas, and six species of Bacillus (Table 4, Fig. 4). However, strains GCM076 and GCM082 were classified as novel, unrecorded species, showing no similarity to any of the strains available in the NCBI database (data not shown).

Table 4.

Identification of isolated strains from various wild type rice cultivars by 16S rDNA sequence analysis

Strain	Homologous microorganism (% identity)	GenBank accession no.
BSG011	Bacillus amyloliquefaciens (100%)	OR342274.1
GCM136	Bacillus amyloliquefaciens (99%)	OR342274.1
GCM190	Bacillus subtilis (100%)	NC_000964.3
GCM219	Bacillus subtilis (99%)	NC_000964.3
HRP003	Bacillus cereus (99%)	NZ_CP072774.1
ICBR02	Serratia liquefaciens (99%)	NZ_CP048784.1
IKBR40	Ewingella americana (99%)	NZ_VXKG01000019.1
IMS042	Bacillus amyloliquefaciencs (99%)	ON287186.1
IMS064	Pseudomonas iranensis (99%)	NZ_LT629788.1
IZGR24	Serratia liquefaciens (99%)	NZ_CP048784.1

Fig. 4.

Phylogenetic tree expressing the relationships of identified bacterial endophytes to taxonomically similar microorganisms based on the 16S rDNA gene sequences. Support values (posterior probability/maximum likelihood bootstrap) are indicated above the branches. Branch lengths are proportional to levels of sequence divergence.

Effect of antagonistic microorganisms on lettuce growth and Sclerotinia rot control

In the pilot experiment, two isolates, Bacillus subtilis GCM190 and an unidentified species of strain GCM082, which controlled Sclerotinia rot by more than 70%, were selected and used for the greenhouse experiment (data not shown). Lettuce treated with the selected antagonistic microorganism Bacillus subtilis GCM190 and the unidentified species of the strain GCM082 had disease control rates of 63.6% and 86.4%, respectively (Fig. 5). With respect to Sclerotinia rot, Bacillus subtilis GCM190 exhibited disease control efficacy comparable to that of the fungicide tebuconazole, which was used as a positive control (Table 5). Previous studies have shown that numerous Bacillus isolates from various sources exhibit antifungal effects against fungal pathogens, such as F. oxysporum, P. capsici, R. solani AG-4, and S. sclerotiorum (Ji et al., 2014; Kim et al., 2008). Similarly, a mixed antagonistic microbial formulation composed of various microorganisms has been reported to effectively suppress cucumber wilt, tomato wilt, spinach damping-off, and Sclerotinia rot (Jung et al., 2005) and the application of Bacillus species to tomatoes has been shown to effectively prevent various plant diseases, including Fusarium spp. and Phytophthora spp. (Koffi et al., 2024). Bacillus subtilis synthesizes cyclic lipopeptides with strong antimicrobial properties, which can be categorized into three groups: surfactins, fengycins, and iturins (Ferreira et al., 2021). These compounds exhibit antibacterial, antifungal, and antiviral activities (Ferreira et al., 2021).

Fig. 5.

Effect of selected antagonistic microorganism on the control of Sclerotinia rot in lettuce. (A) Before pathogen inoculation. (B) At 14 days after pathogen inoculation. GCM082 and GCM190 are bacteria. Tebuconazole: fungicide control. Inoculated: only pathogen inoculated. Healthy denotes noninoculated control.

Table 5.

Effect of two different bacteria on control of Sclerotinia rot in pot experiment

Treatment	Concentration	Mortality (%)
		Mar 14	Mar 28
GCM082	0.1 O.D.	0 aa	20.0 b
GCM190	0.1 O.D.	0 a	7.5 a
Tebuconazole	0.5 mL/L	0 a	5.0 a
Pathogen only	-	0 a	55.0 c
Healty	-	0 a	0 a

^aMeans followed by the same letters are not significantly different within the column a P = 0.05 (LSD).

Healthy is noninoculated. Tebuconazole is a positive control for two bacteria. Pathogen only means the Sclerotinia inoculated.

Bacillus species produce various antifungal compounds, including lipopeptides, such as iturin A and surfactin, which disrupt fungal cell membranes and inhibit their growth (Chen et al., 2015; Wang et al., 2020). Antimicrobial agents produced by Bacillus strains are noncytotoxic, indicating their potential for safe use (Ye et al., 2023). Bacillus species from the soil, including a novel strain closely related to B. siamensis, have been identified to produce bioactive compounds with antifungal properties (Elamin et al., 2021). These compounds have shown effectiveness against zygomycete fungi, surpassing that of standard antifungal agents such as amphotericin B (Elamin et al., 2021).

The number and size of all lettuce leaves used for the determination of plant growth-promoting effects did not show significant differences, regardless of the treatment with the selected antagonistic microorganism Bacillus subtilis GCM190 and the unidentified species of the strain GCM082, as shown in Table 6. However, when treated Bacillus subtilis GCM190 and the unidentified species of strain GCM082, the dry weight of lettuce leaves increased by up to 31%, stems by up to 37%, and roots by up to 7% compared to the untreated control (Table 6). Visual assessment (data not shown) showed that the root growth of lettuce treated with the selected antagonistic microorganism Bacillus subtilis GCM190 and the unidentified species of strain GCM082 was significantly better than that of the untreated control (Fig. 6). Treatment with antagonistic microorganisms improves nutrient availability and soil structure (Normaniza et al., 2018), enhancing root development and overall plant vigor and growth in tree seedlings (Wang et al., 2015), while having no significant effect on the total number of leaves in the plants of pepper and apple (Da Silva et al., 2006; Konovalov et al., 2024), which is in agreement with previous reports. In terms of chlorophyll content, the Bacillus subtilis GCM190 and the unidentified species of the strain GCM082 treatment groups consistently showed higher values than did the untreated control throughout the experiment, with up to a 13% increase observed when treated twice at 1-week intervals (Table 7). Treatment with Bacillus spp. has been reported to improve photosynthetic pigment content and nutrient absorption in apple plants, supporting overall plant health and productivity (Konovalov et al., 2024). Treatment with Bacillus strains, such as B. cereus, B. subtilis, and a mixture of B. amyloliquefaciens increases the maximum photochemical quantum yield of photosystem II, improving the chlorophyll fluorescence and gas exchange parameters (Samaniego-Gámez et al., 2016). Additionally, Bacillus species can support chlorophyll synthesis by solubilizing essential nutrients, such as phosphorus, and producing growth hormones, such as IAA, which promote root development and nutrient absorption (Kulkova et al., 2023).

Table 6.

Effect of antagonistic microorganisms on the growth promotion of lettuce

Treatment	Concentration (O.D.)	Leaf number (no.)a	Dry weight (g)
			Leafb	Stemc	Rootd
GCM082	0.1	145 ae	5.0 a	0.86 a	14.1 ab
GCM190	0.1	149 a	5.5 a	0.81 a	14.7 a
Control (healthy)	-	149 a	4.2 b	0.63 b	13.8 b

^aThe number of all lettuce leaves larger than 10 mm in length attached at two weeks after the third bacterial treatment.

^bThe total dry weight of lettuce leaves (all leaves from 10 pots per replicate) after two weeks of the third treatment.

^cThe total dry weight of stem (all stems from 10 pots per replicate) after two weeks of the third treatment.

^dThe total dry weight of root (all roots from 10 pots per replicate) after two weeks of the third treatment.

^eMeans followed by the same letters are not significantly different within the column at P = 0.05 (least significant difference).

Fig. 6.

Lettuce roots by treatment, 2 weeks after the third application of selected antagonistic microorganisms.

Table 7.

Effect of antagonistic microorganisms on the chlorophyll index of lettuce grown in pots

Treatment	Concentration (O.D.)	Chlorophyll index
		Mar 14a	Mar 21	Mar 28	Apr 4	Apr 10
GCM082	0.1	15.5 ab	20.3 a	19.4 a	22.3 a	24.4 a
GCM190	0.1	15.4 a	20.2 a	19.4 a	22.0 a	24.8 a
Control	-	15.1 a	19.0 b	18.6 a	19.7 b	23.2 b

^aChlorophyll index for one week after antagonistic microorganisms treatment. antagonistic microorganisms treatment was performed on May 14, 21, and 28 at 1 PM.

^bMeans followed by the same letters are not significantly different within the column at P = 0.05 (least significant difference).

The same amount of water (30 mL/pot) was applied across all treatments; however, lettuce plants without the application of the selected antagonistic microorganisms exhibited signs of water deficiency compared to those treated with Bacillus subtilis GCM190 and the unidentified species of the strain GCM082. PGPB strains belonging to the genera Pseudomonas and Bacillus have been reported to promote growth and induce resistance to water stress in mung beans (Saravanakumar et al., 2011). Additionally, the use of Bacillus has been reported to significantly reduce the damage caused by root-knot nematodes in tomato plants, resulting in a 46.3% reduction in root gall formation and a 78.31% decrease in the number of egg sacs in pot experiments, whereas field trials showed a 17.02% increase in fruit yield and an 11.85% increase in harvest per plant after 120 days (Geng, 2024). These Bacillus strains act as effective biofertilizers, promote plant growth, and enhance disease resistance in tomatoes (Koffi et al., 2024). Another study has shown that Bacillus strains produce antimicrobial substances that are effective against various pathogens, including E. coli and S. aureus (Basi-Chipalu et al., 2023). This indicates that the use of antagonistic microorganisms belonging to the Bacillus spp. is effective for crop cultivation and has the potential to positively affect human health.

Taken together, the results demonstrate that antagonistic microorganisms can provide effective disease control, and PGPR effects. The application of antagonistic microorganisms such as Bacillus subtilis GCM190 and an unidentified species of strain GCM082 could act as effective biofertilizers that promote plant growth and enhance disease resistance in various plants, including lettuce, with promising potential for broad agricultural applications.