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A total of six microbial strains were used in this study. Pseudomonas fluorescens KACC12332 and Bacillus subtilis KACC13751 were obtained from the National Agrobiodiversity Center, RDA, Korea. Three additional Bacillus strains (Bacillus 1, Bacillus 2, and Bacillus 3) were provided by the Plant Functional Genomics Laboratory at Pusan National University. Escherichia coli was used as a non-plant-growth-promoting control.

P. fluorescens was cultured in Tryptic Soy Broth (TSB; Merck KGaA, Darmstadt, Germany), while Bacillus spp. and E. coli were cultured in Nutrient Broth (NB; Merck KGaA). Each strain was inoculated at 2 mL into 250 mL Erlenmeyer flasks containing 100 mL of broth and incubated at 30°C for 24 h in a shaking incubator (Lab Companion, Korea) at 150 rpm. The optical density (OD₆₀₀) of cultures was measured hourly using a spectrophotometer (UV-1800, Shimadzu, Japan) to determine the exponential growth phase. Viable cell count was determined by serial 10-fold dilution and plating on respective agar media using the spread plate method to calculate colony-forming units (CFU/mL).

The culture at the peak of exponential growth was defined as the "standard concentration" (OD₆₀₀ ≈ 1.0), and three additional dilutions (1/2, 1/4, and 1/8) were prepared in sterile distilled water to create four concentration treatments for each microbial strain.

Seeds of watermelon (Citrullus lanatus) cultivar ‘Hwalgichan’ (Hyundai Seed Co., Icheon, Korea) were used in this experiment. For each treatment, 10 g of seeds were immersed in 50 mL of the microbial suspension (prepared as above) supplemented with 1% polyvinyl alcohol (PVA) as an adhesive. Seeds were soaked at 30°C for 24 h. After treatment, the seeds were rinsed three times with sterile distilled water and air-dried at 25°C for 12 h prior to use in germination tests.

Germination performance and seedling vigor were assessed using both standard germination and between paper (BP) methods. For the standard germination test, 50 seeds per treatment were placed in sterile 90 mm Petridishes lined with two layers of Whatman No. 2 filter paper. The experiment was arranged in a randomized complete design (RCD) with three replicates per treatment. Petridishes were incubated at 25°C in the dark, and germination was recorded daily for 14 days. Final germination percentage was calculated based on the number of seeds showing radicle emergence. The time to 50% germination (T₅₀) was calculated using the method of Coolbear et al. (1984).

For the BP test, 50 seeds were placed at uniform intervals on heavy germination paper, rolled, and incubated vertically at 25°C in the dark. On days 5 and 14, seedlings were evaluated and classified as normal, abnormal, or ungerminated. Normal seedlings were defined as those with well-developed roots and shoots without morphological abnormalities, while seedlings with damaged or malformed organs were considered abnormal.

Seedling vigor parameters were measured on day 5. Hypocotyl length was measured from the base of the cotyledons to the seed attachment point, and root length was measured from the root-shoot junction to the tip of the primary root. Fresh weight was recorded immediately, and dry weight was determined after oven-drying at 70°C for 72 h.

After 24 h of incubation, the OD₆₀₀ of each microbial culture was measured using a spectrophotometer (UV-1800, Shimadzu, Japan). Cultures were serially diluted 10-fold and plated on Nutrient Agar (NB) or Tryptic Soy Agar (TSA; Difco Laboratories, Detroit, MI, USA), then incubated at 37°C for 24 h. Colonies were counted to determine CFU/mL. The microbial concentrations for each dilution level (OD₆₀₀ and CFU/mL) are presented in Table 1.

The germination characteristics of watermelon seeds were significantly influenced by both the species of PBMs and their dilution levels (Table 2). In particular, higher microbial concentrations resulted in reduced germination, whereas certain diluted treatments enhanced seed vigor.

Pseudomonas fluorescens (KACC12332) at 1/8 dilution showed the highest germination percentage (100%) and a favorable germination speed (T₅₀ = 2.2 days). In contrast, the standard concentration and 1/4 dilution significantly reduced germination rates to 64.4% and 63.3%, respectively. These inhibitory effects at higher concentrations may be attributed to increased production of organic acids, accumulation of extracellular metabolites, or microbial-induced oxygen depletion during germination (Glick, 2012; Boughalleb-M’Hamdi et al., 2018). Overall, P. fluorescens exhibited a dose-dependent negative effect on germination at elevated concentrations.

Bacillus subtilis (KACC13751) maintained relatively high germination rates (89.2–98.9%) across all concentrations, with T₅₀ values ranging from 2.2 to 3.0 days. Notably, the 1/4 dilution showed improved germination speed, suggesting that B. subtilis does not interfere with seed germination and may promote early emergence at moderate concentrations (Compant et al., 2005).

Among the indigenous strains, Bacillus 1 and Bacillus 2 demonstrated the most favorable results in terms of germination percentage, germination speed, and normal seedling rate. The 1/4 dilution of Bacillus 1, the 1/4 and 1/8 dilutions of Bacillus 2, and the 1/2 dilution of Bacillus 3 all achieved 100% germination and 100% normal seedling rate, with rapid T₅₀ values ranging from 1.7 to 1.8 days. These effects are likely associated with the microbial production of bioactive compounds such as auxins and siderophores, which stimulate seed physiological activity (Lugtenberg and Kamilova, 2009; Glick, 2012).

However, Bacillus 3 at standard concentration resulted in reduced germination, while lower dilutions produced responses similar to the untreated control. This indicates a strong sensitivity of seed response to microbial concentration, emphasizing the importance of optimal dosage (Shen et al., 2023). Conversely, Escherichia coli treatments showed no significant differences from the control across all concentrations, indicating minimal influence on seed germination or seedling emergence.

Results from the BP (between paper) germination test aligned with these findings. Most treatments had normal seedling rates exceeding 90%, with several diluted treatments of Bacillus 1 and Bacillus 3 achieving 100%. Although high-concentration Bacillus treatments slightly suppressed germination, they did not produce abnormal seedlings. This suggests that microbial seed coating may improve stress resistance in watermelon seedlings. Similar results were reported by Shahzad et al.(2017), who observed enhanced germination and salinity tolerance in wheat following seed coating with Bacillus spp.

Collectively, these findings indicate that while high microbial concentrations can hinder germination, appropriately diluted treatments can significantly improve seed vigor. Among the treatments tested, the 1/4 dilution of Bacillus 2 and the 1/2 dilution of Bacillus 3 were the most effective, offering a balanced improvement in germination percentage, speed (T_₅₀), and seedling quality. These strains are found to be reliable biological agents for seed treatment in watermelon production technology.

Seed coating with PBMs has emerged as a sustainable biological technology capable of enhancing physiological vigor during the early growth stages, improving stress tolerance, and suppressing disease incidence in horticultural crops (Glick, 2012; Backer et al., 2018). For fruit vegetables such as watermelon, where seedling establishment is critical for subsequent growth and yield formation, microbial seed treatments can play a vital role in securing early vigor (Lee et al., 2000). These biological interventions are also advantageous for reducing chemical input and enhancing eco-efficiency in crop production.

However, the efficacy of microbial seed treatments is highly dependent on microbial strain, host crop physiology, inoculation dose, and environmental context. Therefore, optimizing strain selection and treatment conditions for each crop is essential for commercial deployment and consistent field performance.

In the present study, seed treatment with beneficial microbes significantly influenced early seedling growth traits, including hypocotyl length and diameter, root length, fresh weight, and dry weight, depending on PBM used and dilution level (Table 3, Fig. 1). Several low-dilution treatments of Bacillus strains significantly promoted seedling growth, likely due to microbial interaction with germinating seeds through the production of plant growth-promoting substances such as auxins and siderophores. These compounds may enhance cellular activity and nutrient uptake by improving the rhizosphere environment.

Fig. 1. 
Effect of microbial species and dilution rates on the early growth of watermelon seedlings evaluated 7 days after sowing. Watermelon seeds were treated by biopriming in microbial suspensions at 30°C for 24 h and then sown. Seedlings were grown at 25°C, and growth parameters were measured 7 days after sowing.

Among the treatments, the 1/2 dilution of Bacillus 3 was most effective, producing the highest root length (7.3 cm), fresh weight (241.3 mg), and dry weight (18.3 mg). The 1/4 dilution of Bacillus 2 also led to superior biomass accumulation (fresh weight: 227.3 mg; dry weight: 19.7 mg). These results suggest that optimally diluted biopriming treatments with Bacillus strains can significantly enhance early seedling vigor. The observed growth-promotion effects due to microbial secretion of indole-3-acetic acid (IAA), siderophores, and volatile organic compounds (VOCs), as well as stable rhizosphere colonization that facilitates nutrient uptake and physiological activation (Lugtenberg and Kamilova, 2009; Glick, 2012; Vurukonda et al., 2016).

In contrast, the undiluted treatment of Pseudomonas fluorescens (KACC12332) significantly inhibited seedling development, particularly hypocotyl and root growth. This suppression may result from excessive accumulation of organic acids, antimicrobial metabolites, or oxygen competition due to high microbial density, factors that may disrupt the balance of the germination microenvironment and induce physiological stress.

Such negative effects of microbial seed treatments are not rare and may be a consequence of microbial strain-specific traits, physiological incompatibility with the host crop, antagonistic microbial interactions, or inefficient delivery through coating matrices (Barbara et al., 2019). Similar findings have been reported previously; diane(2007) noted limited nitrogen fixation effects following inoculation with nitrogen-fixing bacteria, while Kay and Stewart (1994) observed reduced efficacy of biological control treatments compared to chemical controls. Moreover, Diniz et al.(2009) reported that a combination of Trichoderma spp. and Beauveria bassiana reduced germination and seedling height in coated seeds.

These observations reinforce that the effectiveness of microbial seed coating is not only determined by the inherent properties of the microbial strain but also governed by the complex interrelation between microbial traits, host plant responses, treatment dose, and environmental factors. In our study, Bacillus subtilis (KACC13751) had negligible or slightly negative effects on seedling development, suggesting low IAA-producing potential or limited compatibility with watermelon (Backer et al., 2018).

The Escherichia coli treatment showed no significant effects on most growth traits, though minor increases in hypocotyl and root length were observed at some concentrations. This may reflect the absence of plant growth promoting metabolites or hormone-like activity in this non-pathogenic control strain.

Overall, the results demonstrate that microbial seed coating effectiveness is determined by a combination of multiple factors including microbial physiology, host–microbe interaction compatibility, dosage precision, and environmental conditions. Among tested treatments, the 1/2 dilution of Bacillus 3 and the 1/4 dilution of Bacillus 2 proved most effective in enhancing watermelon seedling growth, showing significant improvements in root length, fresh weight, and dry weight. These effects were achieved at optimal microbial densities of approximately 1.0–2.3 × 10⁷ CFU/mL.

Given that seedling establishment is a critical determinant of crop productivity and fruit quality in watermelon (Lee et al., 2000; Yoo et al., 2014), strengthening physiological vigor during the early growth phase is essential for successful cultivation. Thus, microbial biopriming represents a promising strategy to enhance seedling establishment and production stability.

In conclusion, Bacillus 2 and Bacillus 3 at optimized dilution rates not only enhanced germination percentage and seedling vigor but also showed superior compatibility with watermelon physiology. These strains are amenable to large-scale culturing and show high environmental adaptability, making them strong preferences for the development of commercial biostimulant seed coating formulations. Our findings provide strong scientific evidence supporting the crop-specific optimization of microbial seed coating protocols as a foundation for broader adoption of biological seed enhancement technologies in sustainable horticulture.