Agriculture plays a critical role in global food security but is constantly threatened by plant pathogens, which cause significant yield and quality losses (Ali et al., 2016). Fungal pathogens are estimated to cause losses of 10-23% of global crop production annually, resulting in major economic losses and threatening food security (Savary et al., 2019). Current crop protection strategies, however, are controversial as they depend heavily on the extensive use of fungicides in both conventional and organic farming (Beckerman et al., 2023). Moreover, intensive fungicide use can accelerate the evolution of resistant pathogen populations, and resistance has emerged to all major classes of pesticides and antimicrobials (Fisher et al., 2018; Hawkins and Fraaije, 2021). Resistance typically arises when rare mutants survive fungicide exposure and proliferate, often through alterations in the fungicide target site or other resistance mechanisms (Brent and Hollomon, 2007).
Several sustainable strategies have been developed as alternatives to chemical fungicides (Kim et al., 2021). Biological control agents (BCAs) are increasingly used because they can suppress fungal diseases, including postharvest rots, while reducing environmental and health concerns associated with chemical inputs (Droby et al., 2009). BCAs include living organisms or products derived from living organisms (e.g., microorganisms, insects, nematodes, and genetically modified crops) (Panpatte et al., 2016). Interest in biological control has grown rapidly due to the need for environmentally friendly disease management options (Ongena and Jacques, 2008). In addition to disease suppression, many beneficial microbes promote plant growth. Among bacteria, members of Paenibacillus and Pseudomonas are particularly important as soil- and plant-associated biocontrol and plant growth-promoting rhizobacteria (Fira et al., 2018; Rastogi et al., 2026).
Paenibacillus spp. are metabolically versatile bacteria capable of utilizing a wide range of organic and inorganic substrates, which support their survival under diverse environmental conditions (Grady et al., 2016). Many species within this genus exhibit plant growth-promoting traits, including nitrogen fixation, phosphate and potassium solubilization, siderophore production, phytohormone production, and degradation of complex polysaccharides (Pandey et al., 2023). Owing to these characteristics, Paenibacillus has attracted considerable attention as a potential biological control agent. Microbial biological control agents can suppress plant diseases through a variety of mechanisms, including antibiosis, competition for nutrients and niche space, production of lytic enzymes, and induction of host defense responses (Köhl et al., 2019). In addition, some beneficial microorganisms can enhance host resistance without directly interacting with the pathogen, thereby contributing to disease suppression through defense priming or induced resistance (Conrath et al., 2015).
Pseudomonas spp. also possesses broad metabolic adaptability, allowing them to colonize a wide range of ecological niches associated with plants and soil (Panpatte et al., 2016). Members of this genus are functionally diverse and have been widely studied as plant growth-promoting rhizobacteria and biological control agents. Their biocontrol activity is generally associated with multiple mechanisms, including competition for nutrients and colonization sites, antibiotic production, and induction of systemic resistance in plants (Panpatte et al., 2016). Pseudomonas spp. are known to produce diverse antimicrobial metabolites, such as 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, and hydrogen cyanide, which contribute to the suppression of plant pathogens (Raaijmakers et al., 2002). In addition to disease suppression, Pseudomonas spp. can improve plant growth and vigor by protecting plants from pathogen pressure during different stages of development (Panpatte et al., 2016).
The field performance of single biological control agents is often inconsistent because microbial survival and activity are strongly influenced by environmental conditions, host physiology, and interactions with resident microbiota. For this reason, the co-application of compatible biological control agents has been proposed as a strategy to improve efficacy and stability by combining complementary functional traits, such as antibiosis, resource competition, and stimulation of host defense responses (Jia et al., 2023; Minchev et al., 2021; Xu et al., 2011). However, co-application does not necessarily result in improved performance, because antagonistic interactions or niche overlap between partner strains may reduce establishment and effectiveness. Therefore, compatibility and functional complementarity between candidate strains should be experimentally validated before practical application (Xu et al., 2011). Collectively, these considerations support the evaluation of co-applied antagonistic bacteria as a potential strategy for achieving more reliable and sustainable disease control under greenhouse and field conditions (Ding et al., 2024; Xu et al., 2011).
In this study, representative strains of Paenibacillus and Pseudomonas were selected through in vitro screening and evaluated for their antagonistic activity against pepper anthracnose and their plant growth-promoting potential. We further investigated whether co-application of the selected strains could improve disease suppression and plant performance, thereby supporting their potential use as a beneficial microbial consortium for sustainable pepper production.
Materials and Methods
Selection of representative antagonistic Paenibacillus strains by dual-culture assays
A total of 92 Paenibacillus strains from the laboratory collection were tested for their in vitro antagonistic activity against the Colletotrichum siamense GYUN-10096, using a dual culture plate assay. Screened Paenibacillus strains were cultured on tryptic soy agar (TSA) and incubated at 28°C for 48 h. GYUN-10096 was cultured potato dextrose agar (PDA) at 25°C for 7 days, and mycelial plug was cultured for dual culture plate assay. Mycelial plugs (3 mm diameter) were placed 30 mm from the center of peptone-supplemented PDA (PDK) plates. Each bacterial strain was streaked on the opposite side of the plate, and plates were incubated at 25°C. Plates inoculated with the pathogen alone served as the untreated control. The initial screening was used only to identify candidate strains for further evaluation. Then, the 10 strains showing the highest inhibition rates were subjected to a secondary antagonism assay.
Selection of enhanced efficacy by co-application bacterial strains by PGPR assays
Selected strains, including GYUN-2285, were first screened by dual-culture assays to identify isolates with strong antimicrobial activity. To complement the primary strain and construct combinations with potentially complementary functions, additional candidate strains were evaluated for plant growth-promoting (PGP)-related traits, and a compatible partner strain showing complementary plant growth-promoting and antagonistic traits activity was selected for subsequent experiments. Unless otherwise stated, experiments included single-strain treatments and co-application with GYUN-2285.
Additional candidate strains (Supplementary Table 1) were evaluated for PGP activity using a seedling growth assay. Cucumber seeds (cv. Backdadagi) were surface sterilized with 1% NaOCl for 5 min, rinsed twice with SDW, and air-dried. Seeds were soaked in each treatment suspension for 1 h. Then, treated seeds were placed at 1 cm interval on wet towel inside 125 × 125 × 20 mm square dishes to maintain humidity and incubated at 20°C for 4 days, after incubation, the shoot length of the seedlings was measured. SDW was control treatment. All treatment was replicated three times.
Finally, to assess compatibility, GYUN-2285 and candidate partner strains were co-cultured on TSA medium and examined for obvious growth inhibition. Strains showing no obvious inhibition when grown together with GYUN-2285 were considered compatible under the tested conditions.
Broad-spectrum antagonistic activity of selected strains against plant pathogens
To evaluate broad-spectrum antagonistic activity, GYUN-2285 and GYUN-2679 were tested against additional plant-pathogenic fungi using the dual-culture assay described above. The tested pathogens were included Colletotrichum spp. causing apple bitter rot (C. fructicola, C. gloeosporioides, and C. siamense), Diplodia seriata (apple black rot), Botryosphaeria dothidea (apple white rot), Valsa mali (apple valsa canker), Botrytis cinerea (ginseng gray mold), Fusarium solani (ginseng root rot), Colletotrichum spp. causing pepper anthracnose (C. acutatum, C. coccodes, and C. scovillei), and Fusarium oxysporum f. sp. lycopersici (tomato fusarium wilt). Details of isolates are provided in Supplementary Table 2.
Effect of GYUN-2285 and GYUN-2679 on conidial germination of Colletotrichum acutatum
Conidial germination of C. acutatum was evaluated on glass slides treated with GYUN-2285, GYUN-2679, or their mixture. Bacterial suspension were diluted to 106 CFU/mL. For the mixture treatment, suspensions of GYUN-2285 and GYUN-2679 were prepared separately and mixed at a 1:1 (v/v) ratio immediately before application to obtain a final concentration of 1 × 106 CFU/mL. Conidia were harvested from 10-day-old PDA cultures and adjusted to 1 × 105 conidia/mL. Equal volumes (10 μL each) of conidial suspension and bacterial suspension were mixed on the slide surface. A conidial suspension mixed with sterile distilled water served as the control. Slides were incubated at 25°C, and conidial germination was assessed after 0, 8, 16, and 24 h under a light microscope (Colibri 7; Carl Zeiss Microscopy GmbH, Jena, Germany). All treatments were replicated three times. The conidia germination rate (%) was as follows.
Control of pepper anthracnose by GYUN-2285 and GYUN-2679 using in planta assays
To evaluate the efficacy of GYUN-2285, GYUN-2679, and their mixture against pepper anthracnose under in planta conditions, pepper fruits were surface-sterilized with 1% NaOCl for 1 min, rinsed twice with sterile distilled water, and air-dried. Each fruit was wounded with a sterile needle. Bacterial suspensions (1 × 106 CFU/mL) were sprayed onto the fruit surface, and fruits were air-dried. After 12 h, each wound was inoculated with 10 μL of a conidial suspension of C. acutatum (1 × 105 conidia/mL). Fruits treated with sterile distilled water served as the control. All fruits were incubated in humid plastic boxes at 25°C. Each treatment included 12 replicate fruits, and the experiment was conducted twice. Disease severity and control efficacy were evaluated 7 days after inoculation. The disease index scale was established from 0 to 4, where 0 = no symptoms, 1 = disease lesions ≥ 2 mm; 2 = tissue showing disease lesions ≥ 4 mm; 3 = disease lesion < 8 mm; 4 = sunken lesions and spore production. The disease severity (%) and control efficacy (%) are calculated using the following formulae:
Growth promotion effect of GYUN-2285 and GYUN-2679
To assess the growth-promoting potential of GYUN-2285, GYUN-2679, and mixture, the pepper seeds (cv. Color King) were planted in plastic trays (6 × 6 pots per tray) containing garden soil (Nongwoo Bio., Ltd., Yeoju, Korea). After germination, 1-month-old pepper seedlings were soil drenched with 20 mL of bacterial suspensions prepared from 48 h cultures and diluted to 1 × 105, 106, 107, and 108 CFU/mL for each strain. The concentration used for the mixture treatment was selected based on the most effective single-strain treatment. Seedlings were maintained in a greenhouse at 25 ± 5°C with a 12-h light/12-h dark photoperiod. Treatments were applied three times at 7-day intervals, and growth was recorded 7 days after the final application. Each treatment included 15 replicate seedlings, and SDW was used as the control.
Colonization and persistence of GYUN-2285 and GYUN-2679 on pepper fruit surfaces
To evaluate changes in the surface persistence of GYUN-2285 and GYUN-2679 on pepper fruits over time, pepper fruits were surface sterilized in 1% NaOCl for 1 min, rinsed twice with SDW, and air-dried. Bacteria suspension (1 × 107 CFU/mL) were prepared culturing each strain on TSA for 48 h, harvesting cells, and adjusting the concentration with SDW. The suspensions were sprayed onto the surface using atomizer for 20 mL each treatment and control was treated with SDW. After air-drying, fruits were placed in humid plastic trays and incubated at 28°C. Surface samples were collected every 2 days from day 0 to 16 days. For each sampling time, a cut into 10 × 10 mm diameter plant tissue was excised from each fruit, homogenized in 10 ml SDW using a sterile mortar and pestle, and serially diluted (10-fold). Each 100 μL of appropriate dilutions were spread on TSA plates and incubated at 28°C for 48 h to determine CFU. Three fruits were used per treatment at each sampling time.
Field evaluation of GYUN-2285 and GYUN-2679 for suppression of pepper anthracnose
A field trial was conducted to evaluate the efficacy of GYUN-2285, GYUN-2679, and their mixture for the control of pepper anthracnose. Pepper seedlings (cv. Color King) were transplanted into a field located at Gyeongkuk National University farm (36°32′42.72″N, 128°48′6.84″E). Plants were spaced 800 mm between rows and 350 mm within rows. Treatments included untreated control, a systemic chemical control schedule, two single chemical treatments (tebuconazole, 25% WP; pyraclostrobin, 22.9% EC), the registered biocontrol agent Bacillus velezensis AK-0 used for pepper anthracnose, GYUN-2285, GYUN-2679, and the mixture. The field trial was arranged in a randomized complete block design with three replicate plots per treatment, and each plot consisted of 20 plants. No artificial inoculation of the pathogen was performed, and disease development was evaluated under natural infection conditions. All bacterial treatments were applied as foliar sprays at 1 × 106 CFU/mL, with 250 mL of bacterial suspension applied per plant. Applications were initiated on July 12, 2024, and repeated five times at 10-day intervals until August 23, 2024, using a foliar spray method (the detailed treatment plan is shown in Supplementary Table 3). Fruits were harvested 10 days after the final application, and control efficacy was calculated as described above.
Statistical analysis
Data were analyzed by analysis of variance (ANOVA) using R (R Foundation for Statistical Computing, Vienna, Austria). For the field experiment, treatment means from three replicate plots were subjected to ANOVA. When ANOVA indicated significant treatment effects, means were separated using Fisher’s least significant difference (LSD) test at α = 0.05.
Results
In vitro screening of antagonistic Paenibacillus and Pseudomonas strains
In the initial screening, candidate strains showing relatively high antagonistic activity were selected for further evaluation (Supplementary Table 4). The top 10 strains were subjected to a secondary screening, and among these final candidates, GYUN-2285 showed the strongest and most consistent antagonistic activity and was therefore selected for subsequent experiments (Supplementary Fig. 1).
To identify a co-application partner for GYUN-2285, six candidate strains were evaluated for plant growth-promoting activity and compatibility with GYUN-2285 on the same medium. Among single-strain treatment, GYUN-2285 showed one of the highest shoot lengths (49.53 mm) compared with the SDW control (45.07 mm). In the preliminary seedling screening assay (Supplementary Fig. 2), co-application of GYUN-2285 and GYUN-2679 resulted in the greatest increase in shoot length amoing the tested strain combinations. In addition, GYUN-2679 was able to grow together with GYUN-2285 on TSA without obvious inhibition (Supplementary Fig. 3). Therefore, GYUN-2679 was selected as a co-application partner based on its complementary growth-promoting performance and compatibility with GYUN-2285 under the tested conditions.
In vitro antagonistic activity of GYUN-2285 and GYUN-2679
The in vitro antifungal activities of GYUN-2285 and GYUN-2679 were evaluated against 12 fungal plant pathogen (Fig. 1). Overall, GYUN-2285 showed stronger inhibition of mycelial growth than GYUN-2679 across most of the test pathogens. In particular, GYUN-2285 exhibited relatively strong inhibition activity against several Colletotrichum species, including C. acutatum, C. coccodes, and C. scovillei, which are associated with pepper anthracnose (Fig. 1A). GYUN-2679 also showed inhibitory activity, although its overall antifungal effect was generally weaker than that of GYUN-2285 (Fig. 1B). Based on these results, C. acutatum was selected as the target pathogen for subsequent conidial germination and in plant assays.
Inhibition of conidia germination effect for Colletotrichum acutatum
Both bacterial treatments reduced conidial germination relative to the control, and the mixed treatment showed the strongest inhibitory effect. The control group exhibited 100% germination after 24 h, while the GYUN-2285 treated exhibited 31.3%, GYUN-2679 treated exhibited 0%, and mixture of GYUN-2285 and GYUN-2679 exhibited 0% germination, respectively (Fig. 2). Under all treatment, appressorium formation was not observed.
Control of pepper anthracnose by GYUN-2285 and GYUN-2679
Pepper fruits were wounded prior to inoculation to ensure uniform infection and allow consistent comparison of treatment effects under controlled conditions. The mixture of GYUN-2285 and GYUN-2679 demonstrated a control value of 88% against C. acutatum in the wounded inoculation, whereas each single treat attained a control value of 59% and 67% (Fig. 3). In wounded pepper-fruit assays, both single-strain treatments significantly reduced anthracnose incidence compared with the control, and the combined treatment showed the highest control value (Fig. 3). These results suggest complementary or additive effects under co-application rather than demonstrating synergy.
Plant growth promoting effect by GYUN-2285 and GYUN-2679
Shoot length increase differed significantly among treatments (Fig. 4). The SDW-treated control showed a shoot length increase of 38.14 ± 3.71 mm. Across the single-strain treatments, GYUN-2679 generally promoted seedling growth more effectively than GYUN-2285. GYUN-2679 at 106 CFU/mL resulted in the highest shoot length increase among single applications (41.69 ± 1.17 mm), followed by 105 CFU/mL (39.60 ± 4.24 mm), whereas growth promotion decreased at 107 (34.47 ± 3.54 mm) and 108 CFU/mL (32.33 ± 3.70 mm). In contrast, GYUN-2285 alone did not enhance shoot growth across the tested concentrations, showing markedly lower shoot length increases at 105 (17.50 ± 2.53 mm), 106 (26.33 ± 2.68 mm), 107 (16.83 ± 0.91 mm), and 108 CFU/mL (14.83 ± 1.75 mm) compared with the control. Notably, the combined application of GYUN-2285 and GYUN-2679 (1:1 mixture at 106 CFU/mL) produced the greatest numerical increase in shoot length (49.53 ± 4.61 mm) and was significantly greater than the control (Fig. 4).
Colonization and persistence of GYUN-2285 and GYUN-2679 on pepper fruit surfaces
The populations of GYUN-2285 and GYUN-2679 on pepper fruit surfaces declined over time after application (Fig. 5). At day 0, both strains were recovered at comparable levels, with 6.1 × 104 CFU per sampled tissue piece for GYUN-2285 and 6.4 × 104 CFU per sampled tissue piece for GYUN-2679. Thereafter, GYUN-2285 declined moderately and remained relatively stable throughout the observation period, fluctuating between 3.8 × 104 and 5.4 × 104 CFU per sampled tissue piece from 2 to 16 days after application. In contrast, GYUN-2679 showed a more pronounced decline, decreasing to approximately 2.1 × 104 CFU per sampled tissue piece by day 14. Overall, both strains persisted on pepper fruit surfaces for at least two weeks after treatment, with GYUN-2285 maintaining higher population levels than GYUN-2679 at later sampling times (Fig. 5).
Effect on disease control of pepper anthracnose under field conditions
Under field conditions, the mixture showed a control value of 47.3%, compared with 42.8% for GYUN-2285 and 30.6% for GYUN-2679. The mixture showed numerically higher anthracnose suppression than the single-strain treatments and performance comparable to the better-performing reference treatments, including the systemic chemical treatment (52.2%) and AK-0 (53.4%), under the conditions tested (Fig. 6).
Discussion
Anthracnose is one of the most destructive diseases of pepper, causing substantial losses in fruit yield and quality (Han et al., 2025). In this study, to evaluated whether co-application of P. polymyxa GYUN-2285 and P. protegens GYUN-2679 could improve disease suppression and plant growth relative to single-strain treatments. Although both genera are well known as biological control and plant growth-promoting bacteria, relatively few studies have examined their combined application in the pepper anthracnose pathosystem under both controlled and field conditions. Our results indicate that co-application of these two strains improved overall performance under the tested conditions, although the observed effects should not be interpreted as definitive evidence of synergy.
The functional complementarity of the two strains across infection stages of Colletotrichum. Dual culture assays showed that GYUN-2285 exerted stronger inhibition of mycelial growth than GYUN-2679 against a broad panel of fungal pathogens, including several Colletotrichum spp. (Fig. 1). In contrast, the conidial germination assay indicated that GYUN-2679 strongly suppressed germination, reaching complete inhibition at 24 h, whereas GYUN-2285 alone showed partial suppression (Fig. 2). The combined treatment resulted in higher numerical suppression than single strain treatment under the conditions tested. Because formal interaction analyses were not performed, the observed effect should not be interpreted as definitive evidence of synergy. Instead, the results suggest possible functional complementarity under the tested conditions.
The enhanced disease suppression observed in planta is consistent with improved efficacy under co-application (Price-Christenson and Yannarell, 2023). In wounded pepper-fruit assays, the mixture achieved substantially higher control efficacy than the individual treatments (Fig. 3). The combined treatment resulted in higher numerical suppression than either single-strain treatment under the conditions tested. These results suggest possible functional complementarity under the tested conditions (Nguyen et al., 2025). Wound inoculation bypasses natural penetration processes, these results should be interpreted as controlled comparative data rather than a direct simulation of natural infection. Nevertheless, the fruit assay provides useful evidence that co-application can improve disease suppression under a standardized infection system.
Although the mixture treatment showed the greatest numerical increase in shoot length, the single-strain results indicate that the two bacteria did not contribute equally to plant growth promotion. GYUN-2285 alone did not consistently enhance shoot growth and, at some concentrations, showed lower shoot elongation than the control. This suggests that GYUN-2285 contributed primarily through antagonistic activity rather than direct growth promotion. In contrast, GYUN-2679 showed a clearer growth-promoting effect across the tested conditions, indicating that it contributed more strongly to seedling growth promotion. Therefore, the improved performance of the combined treatment is best interpreted as the result of combining different functional traits of the two strains, rather than assuming that both strains independently promoted plant growth to a similar extent.
Persistence on host surfaces is another important factor influencing the field performance of microbial biological control agents. In this study, recoverable populations of both strains remained detectable on pepper fruit surfaces for at least two weeks after application, although GYUN-2285 maintained higher populations than GYUN-2679 at later sampling times. This difference may partly explain why GYUN-2285 contributed more strongly to sustained suppression after application, whereas GYUN-2679 may have contributed more strongly to early-stage inhibition. At the same time, because strain-specific identification was not performed in the surface persistence assay, these results should be interpreted cautiously as colony recovery on TSA rather than definitive confirmation of the applied strains alone. Nevertheless, the persistence data suggest that both strains were capable of remaining on the fruit surface long enough to contribute to disease suppression after application. This point is relevant because persistence and environmental stability are often major determinants of field reliability for microbial sprays.
The field application provided additional support for the practical relevance of the co-application strategy. Under natural infection conditions, the mixture showed numerically higher anthracnose suppression than either single-strain treatment and performed comparably to the better performing reference treatments under the tested conditions. Because this field trial was conducted at a single location in a single season, these results should be interpreted as preliminary field validation rather than definitive evidence of broad field stability. Field performance of microbial biological control agents is strongly influenced by environmental variability and interactions with resident microbiota (Bardin et al., 2015). Therefore, although the numerically improved field performance of the mixture may reflect the contribution of complementary traits under variable conditions, additional multi-location and multi season trials will be necessary before broader practical recommendation.
Possible mechanisms underlying the observed effects were not directly investigated in this study. Therefore, mechanisms such as antibiosis, competition for nutrients and niche space, host defense induction, or enzyme-mediated antagonism should be regarded as plausible explanations based on previous studies of Paenibacillus and Pseudomonas, rather than as mechanisms demonstrated here. In conclusion, the present study shows that co-application of P. polymyxa GYUN-2285 and P. protegens GYUN-2679 improved overall performance in pepper anthracnose management under the tested conditions. GYUN-2285 was more strongly associated with inhibition of mycelial growth, whereas GYUN-2679 contributed more strongly to conidial germination suppression and seedling growth promotion. Although additional mechanistic studies and broader field validation are still needed, these results support the potential of this bacterial combination as a candidate biocontrol treatment for pepper anthracnose.
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