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Article

Synergistic Effects of Salicylic Acid and Bacillus butanolivorans KJ40 for Enhancing Napa Cabbage (Brassica napa subsp. pekinensis) Resilience to Water-Deficit Stress

by
Sang Tae Kim
† and
Mee Kyung Sang
*,†
Division of Agricultural Microbiology, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(6), 618; https://doi.org/10.3390/horticulturae10060618
Submission received: 17 May 2024 / Revised: 4 June 2024 / Accepted: 7 June 2024 / Published: 10 June 2024
(This article belongs to the Special Issue Biostimulants and Plant Elicitors to Mitigate the Effect of Biotic and Abiotic Stress, 2nd Edition)
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Abstract

:
Climate change exacerbates drought, globally impacting crop production and necessitating the adoption of sustainable strategies. This study investigates the potential synergistic effects of salicylic acid (SA) and Bacillus butanolivorans KJ40 (KJ40) on napa cabbage (Brassica rapa subsp. pekinensis) under water-deficit stress conditions by watering withheld for five days. Results demonstrate that the combined application of KJ40 and SA, particularly at concentrations of 0.5 mM and 1 mM, significantly enhances plant growth and mitigates the negative impacts of water deficit. Moreover, the combination treatment with SA (0.5 mM) and KJ40 (1 × 108 cells/mL) reduces lipid oxidation and enhances antioxidant enzyme activity, indicating improved plant stress tolerance. Analysis of soil microbial profiles reveals alterations in metabolic activity and substrate utilization patterns, suggesting potential changes in rhizosphere dynamics. Additionally, this study examines the impact of SA on KJ40 population dynamics in soil, revealing concentration-dependent effects on bacterial survival. Overall, the combination of KJ40 and SA was effective in mitigating water-deficit stress in napa cabbage. These findings highlight the combination as a novel synergistic strategy to enhance plant resilience to water-deficit stress, offering insights into plant–microbe interactions and soil ecosystem dynamics.

1. Introduction

Climate change causes multiple impacts on agriculture, food security, and human health; drought, one of the chronic changes as a hazard in climate change, increases the adverse impacts on global agricultural crop production [1]. The area affected by drought is approximately 40% of the world’s available land; additionally, the climate change that may lead to extreme temperatures is predicted to cause severe prolonged drought in some areas [2]. The elevating possibility of drought hazard is predicted, and drought risk has been globally reported in corn, maize, and rice cultivation [3,4,5]. Pörtner et al. [1] reported that maize yields will fall by 23% in the 21st century with high greenhouse gas emissions in the food system. As well as cultivated crops, the drought risk is also suggested in field-grown vegetables, including cabbages [6]. Cultivation of cabbage (Brassica rapa L.), which belongs to the crucifer family and is a very popular vegetable around the world, is constrained by abiotic stress such as water-deficit stress as a result of water shortages or an inefficient irrigation system [2,7]. Drought stress induces a set of physiological and biochemical reactions in plants [7], including seed germination and seedling growth inhibition, and oxidative damage caused by the overproduction of reactive oxygen species [8,9].
Regarding plant damage by abiotic stress, including drought, various sustainable and eco-friendly management strategies are studied to satisfy increasing food demands [10]. Verma et al. [11] elucidated that silicon-activated plant protection and stress tolerance mechanisms, including the enhancement of plant development and root morphology, balance of water status, improvement of photosynthetic pigments and efficiency, activation of enzymatic and non-enzymatic activities, balance of reactive oxidative species and plant hormone level, and accumulation of compatible solute. Seed priming with urea and potassium nitrate markedly improves drought tolerance at the germination stage and early seedling growth of Chinese cabbage with the activation of enzymatic antioxidants, including catalase, superoxide dismutase, and peroxidase, and via the accumulation of non-enzymatic antioxidants, such as proline and soluble sugar contents [12]. Similarly, Haghighi et al. [13] demonstrated that a foliar application of an exogenous amino acid fertilizer containing 16 different amino acid constituents promotes growth and nutritional value, including protein, total protein, proline, and glutamic acid, glutamine, and asparagine of cabbage in regularly watered and drought-stressed plants. Biochar applications in soils improve plant growth, photosynthetic activity, nutrient uptake, and modify physiological and biochemical traits in cabbage seedlings under water-deficit conditions [14]. In addition to nonbiological materials, plant biostimulants, which are defined as biological formulations for the improvement of plant health and productivity, have evolved, and they are involved in promoting growth and enhancing plant defenses against various abiotic stresses, including drought [15,16]. Nephali et al. [17] demonstrated that microbial plant biostimulants enhance drought resilience in maize plants by altering drought resistance-related metabolic pathways, including redox homeostasis, strengthening the plant cell wall, osmoregulation, energy production, and membrane remodeling.
Salicylic acid is well established as a tool for increasing plants’ tolerance to drought stress as well as resistance to plant disease [18]. Pandey and Chakraborty [19] demonstrated that 1 mM of SA pretreatment increases chlorophyll, proline, carbohydrate, total phenolic content, as well as some antioxidant enzyme activities, and induces stress-related gene expressions in Black gram under short-term drought stress. Similarly, the exogenous seed treatment with salicylic and succinic acid mitigates drought stress via the accumulation of proline as an osmoregulatory substance and with the improvement in the water usage efficiency in flowering kale [20]. Despite the advantages, generally high concentrations of salicylic acid, depending on the plant species, adversely regulate plant growth and development, such as seed germination, budding, and flowering [18,21]. Thus, to redeem this disadvantage and implement microbial-based biostimulants into agronomic practices, we need to understand the synergistic effect of microorganisms and salicylic acid on induced tolerance to drought stress in napa cabbage. In our previous study, Bacillus butanolivorans KJ40 alleviated drought stress via modulating phenolic compounds and enhancing enzymatic antioxidant activity for scavenging reactive oxygen species in pepper plants [22].
We hypothesize that the combined application of salicylic acid and the biostimulant bacterial strain KJ40 will synergistically enhance the growth and stress tolerance of napa cabbage under water-deficit conditions by inducing physiological changes in plants and soil metabolic potential. The objectives are to determine the concentration of salicylic acid that does not decrease plant growth and elevates the induced tolerance of KJ40 to water-deficit stress in napa cabbage; to investigate the impact of the combined application of salicylic acid and KJ40 on plant physiological changes, including lipid peroxidation and antioxidant enzyme activity, as well as the accumulation of salicylic acid and glucosinolate contents; and to evaluate the influence of the combination on soil microbial profiles and activity as well as the population dynamics of KJ40.

2. Materials and Methods

2.1. Preparation of Bacterial Strain and Salicylic Acid

The bacterial strain Bacillus butanolivorans KJ40, selected as a drought-tolerant inducing bacteria in our previous study [22], was utilized. The strain was cultured on tryptic soy agar (TSA, Difco, Sparks, MD, USA) at 28 °C for 48 h, and a single colony was then incubated in tryptic soy broth (TSB, Difco, USA) at 28 °C and 160 rpm for 48 h. Following incubation, bacterial cells were collected by centrifugation at 6000 rpm for 20 min, and the bacterial suspension was adjusted to an optical density of A600 nm = 0.2, corresponding to 1 × 108 cells/mL. Salicylic acid (sodium salt, S3007, Sigma-Aldrich, USA) was dissolved in sterile distilled water and used at final concentrations of 0.1, 0.5, and 1 mM for plant growth assays.

2.2. Plant Growth Assay

Napa cabbages (‘Bulam No. 3′, Farm Hannong Co., Ltd., Seoul, Republic of Korea) and then transplanted into plastic pots (diameter 12 cm) filled with pasteurized field soil (105 °C for 1 h, twice at one-day intervals) in an experimental greenhouse at the National Institute of Agricultural Sciences in Wanju-gun province, Republic of Korea. Seven days after transplanting, a mixture of KJ40 cell suspension and salicylic acid was drenched into the plants. Seven days later, water-deficit stress was induced by withholding irrigation. One day before the stress treatment, the pots were fully watered (50 mL/pot), and then watering was withheld for five days. At the termination of the stress period (soil moisture ≈ 11%) or unstressed period (soil moisture ≈ 25%), plant fresh weight was measured, and leaves at the 7th- to 10th-leaf stage were sampled at three days after stress treatment for antioxidant enzyme activity and at five days after stress treatment for the evaluation of malondialdehyde (MDA), proline, salicylic acid, and glucosinolate contents using liquid nitrogen, and they were stored at −80 °C. Simultaneously, rhizosphere soil samples from the napa cabbage plants were collected for soil microbial activity assays. In parallel, a well-watered treatment was conducted as an unstressed condition, with 10 mL of water drenched into each pot daily. Plant fresh weight was assessed with 10 biological replicates per treatment for each experiment, repeated three times.

2.3. Contents of Malondialdehyde and Proline, and Antioxidant Enzyme Activity

Leaf MDA content was quantified following the method described by Dhindsa et al. [23]. Leaf tissues (100 mg) were homogenized in 500 µL of 0.1% trichloroacetic acid (TCA) (w/v) and centrifuged for 10 min at 13,000× g at 4 °C. The resulting supernatant (200 µL) was mixed with 600 µL of 0.5% 2-thiobarbituric acid (TBA) in 20% TCA and then incubated at 90 °C for 30 min. The reaction was terminated by placing the samples on ice for 5 min. Absorbance readings at 450, 532, and 600 nm were recorded using a spectrophotometer (Infinite M200 PRO, TECAN, Switzerland), and MDA content was determined based on the method outlined by Bao et al. [24]. Proline content was determined following the protocol outlined by Bates et al. [25]. Leaf samples (100 mg) were homogenized with 1.2 mL of 3% sulfosalicylic acid, and the resulting mixture was centrifuged at 13,000× g for 10 min. Subsequently, 500 µL of the supernatant was transferred and combined with 500 µL of glacial acetic acid, followed by the addition of 1 mL of acid ninhydrin. The mixture was then incubated at 90 °C for 1 h and subsequently cooled on ice. After incubation, 2 mL of toluene was added to the reaction mixture for 2 min, and the absorbance of the upper layer was measured at 520 nm using a spectrophotometer. Proline content was determined by comparing it to a standard curve generated using L-proline. MDA and proline were determined with 5 biological replicates per treatment for each experiment, repeated twice.
Antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT), were determined following the manufacturer’s instructions for the superoxide dismutase assay kit (Item No. 706002, Cayman Chemical Company, Ann Arbor, MI, USA) and catalase assay kit (Item No. 707002, Cayman Chemical Company, Ann Arbor, MI, USA). For SOD activity measurement, 1 g of leaf tissue was homogenized in 5 mL of cold buffer containing 20 mM HEPES with 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose (pH 7.2). After centrifugation at 12,000× g rpm at 4 °C for 15 min, the supernatant was used. For CAT activity determination, 1 g of leaf tissue was homogenized in 5 mL of cold buffer consisting of 50 mM potassium phosphate and 1 mM EDTA (pH 7). Following centrifugation at 12,000× g rpm for 15 min at 4 °C, the supernatant was used. The antioxidant enzyme activities were performed with 5 biological replicates per treatment for each experiment, repeated twice.

2.4. Salicylic Acid and Glucosinolate Analysis

The leaves (7th- to 10th-leaf stage) were pulverized using liquid nitrogen in a mortar and subdivided into tubes, with each tube containing 0.1 g of leaf tissue. Each sample was analyzed for salicylic acid and glucosinolate content. Salicylic acid analysis was conducted according to the method described by Seskar et al. [26] and Jung et al. [27]. Samples were extracted using 90% and 100% methanol, with ο-anisic acid used as an internal standard. The supernatant was dried under vacuum desiccation and resuspended in 100 mM sodium acetate buffer (pH 5.5) containing 40 units of β-glucosidase. The mixture was then incubated at 37 °C for 1 h and 30 min in a water bath. After incubation, an equal volume of 10% trichloroacetic acid was added to stop the reaction. The mixture was then centrifuged at 4000× g rpm for 15 min, and the top phase was collected. The collected sample was dried and resuspended in 0.5 mL of 55% methanol, followed by filtration using a 0.22 μm nylon syringe filter. The high-performance liquid chromatography (HPLC) solvent consisted of water containing 0.5% acetic acid (solvent A) and methanol (solvent B). The gradient conditions were as follows: 0–7.5 min, 70:30 (solvent A: solvent B); 7.5–12 min, 60:40; 12–15 min, 40:60; and >15 min, 70:30. A reverse C18 column (YMC, Japan) was used, and detection was performed at excitation 301 nm and emission 412 nm using a fluorescence detector.
Glucosinolate analysis was performed according to the method described by Mawlong et al. [28]. Leaf powder was extracted with 2 mL of 80% methanol and incubated at 20 °C overnight. After incubation, the sample was centrifuged at 3000× g rpm for 15 min at 4 °C. The extracts (0.5 mL) were diluted with 0.3 mL of distilled water, and 3 mL of 2 mM sodium tetrachloropalladate was added. The mixture was then incubated for 1 h at room temperature. After incubation, the mixture was measured at 425 nm using a spectrophotometer. Glucosinolate content was determined by comparison to a standard curve generated using sinigrin. The contents of salicylic acid and glucosinolate were determined with 5 biological replicates per treatment for each experiment, repeated twice.

2.5. Soil Microbial Physiological Profile Using EcoPlate and Activity

For the ecoplate assay, soil samples were prepared according to the manufacturer’s instructions (Biolog, Catalog #1506, USA). Ecoplates were measured every 24 h for 72 h using a microplate reader (Infinite M200 Pro, Bionics). For analysis, the optical density (OD) at 590 nm at 72 h was used. For soil microbial activity, including fluorescein diacetate hydrolysis (FDase) and β-glucosidase, root-adhering soil samples of napa cabbages were collected 5 days after water-deficit stress or under unstressed conditions. The FDase was measured as described by Schnurer and Rosswall [29]. Soil samples (1 g) were added to 4 mL of 60 mM sodium phosphate buffer (pH 7.6) containing fluorescein diacetate (FDA) at a final concentration of 10 µg/mL, followed by incubation at 25 °C for 1 h. The reaction was terminated by adding acetone and filtered through a two-layer filter paper (Whatman, No. 2). The filtered solution was then measured at OD 490 nm. The β-glucosidase activity was measured using a soil β-glucosidase (S-β-GC) activity assay kit (BC0160, Solarbio). The ecoplate assay and soil microbial activity were determined with 5 biological replicates per treatment for each experiment, repeated twice.

2.6. Effect of Salicylic Acid on Growth and Colonization of KJ40

To assess the effect of salicylic acid on KJ40′s growth in a medium, filtered salicylic acid (final concentration = 0.5 mM, pore size 0.22 µm) was added to tryptic soy broth (TSB) and inoculated with the KJ40 suspension (A600 = 0.2, 1 × 108 cells/mL) as a mixture (salicylic acid solution/bacterial suspension = 9:1, v/v). Bacterial growth was monitored using Bioscreen C (Thermo Labsystems, USA) for 36 h. For rhizosphere colonization, a spontaneous rifampicin mutant of KJ40 was prepared following the method of Sang and Kim [30]. The bacterial treatment was conducted under the same conditions as described above for the plant assay. Rhizosphere soil samples were collected 1, 4, and 7 days after treatment. One gram of each root-adhering rhizosphere soil sample was collected and serially diluted with 10 mM of the MgSO4 solution. A 100 µL aliquot of the diluted suspension was spread on Tryptic Soy Agar (TSA) amended with rifampicin (50 µg/mL). Colony-Forming Units (CFUs) were counted 72 h after incubation at 28 °C. The bacterial population was measured with 4 biological replicates per treatment for each experiment, repeated twice.

2.7. Statistical Analysis

The data were analyzed and visualized using Python (version 3.9). Repeated experiments’ data were pooled after confirming the homogeneity of variances using Levene’s test. Following verification of normal distribution by the Shapiro–Wilk test, significant differences between each treatment were analyzed using the t-test.

3. Results and Discussion

3.1. Synergistic Effects of Salicylic Acid and Bacillus Butanolivorans KJ40 (KJ40) on Plant Growth under Water-Deficit Stress

To determine the concentration of salicylic acid that does not decrease plant growth and elevates the induced tolerance of KJ40 to water-deficit stress in napa cabbage, 0 (salicylic acid 0 mM, control), 0.1, 0.5, and 1 mM of salicylic acid were drenched either in isolation or in combination with KJ40′s cell suspension. After seven days, non-irrigation for five days was conducted as water-deficit stress (Figure 1). Under unstressed conditions, a single KJ40 application and a combination of KJ40 with SA 0.1 mM significantly increased the fresh weight of napa cabbage compared to the control at p < 0.05 and p < 0.01, respectively (Figure 1). Compared to unstressed conditions, water-deficit stress induced a significant decrease in fresh weight (df = 2, F = 191.0922, p < 0.0001). However, combining KJ40 with SA 0.1 or 0.5 mM significantly enhanced fresh weight compared to the control (Figure 1). Single applications, including KJ40, SA 0.1 mM, and SA 0.5 mM, did not exhibit growth increases compared to the control (Figure 1); therefore, combining KJ40 with SA (0.5 and 1.0 mM) could synergistically impact the promotion of growth under water-deficit conditions. Hence, 0.5 mM of salicylic acid was used as the isolated ‘SA’ and a combination of KJ40 as ‘KSA’, which were used for further study.
Salicylic acid is a key plant hormone involved in plant responses, specifically regarding upholding plant defenses against infections with plant pathogens through the initiation of pathogenesis-related gene expression and defense compound synthesis; tolerance to abiotic stresses such as drought, salt, and heavy metal stresses via a crosstalk with other plant hormones such as abscisic acid; and plant growth [18,31]. Salicylic acid is involved in plant growth by mediating cell division and expansion, and it affects plant growth, depending on its concentration and host species [32]. Furthermore, due to the results of discrepant growth in one plant species affected by salicylic acid [32], it is necessary to determine the threshold of application concentration of salicylic acid according to plant species and development. In our system, napa cabbage (‘Bulam No. 3′, Farm Hannong Co., Ltd., Republic of Korea) did not decrease in fresh weight under a 1.0 mM concentration, and an isolated treatment of KJ40 and in combination with 0.1 mM of salicylic acid showed growth-promoting activity under well-watered conditions. Under water-deficit conditions, combining KJ40 with 0.5 or 1 mM of salicylic acid resulted in a lower decrease in plant growth compared to the control. Interestingly, strain KJ40, which displays plant growth-promoting activity under well-watered conditions, could promote the fresh weight of napa cabbage when combined with salicylic acid (0.5 and 1 mM) under water-deficit conditions. Our previous study [22] demonstrated that the KJ40 strain could induce tolerance to drought stress in pepper plants by regulating the composition of plant phenolic compounds; however, the single KJ40 treatment did not alleviate water-deficit stress in napa cabbages. These results indicate that the KJ40 strain specifically acts to mitigate stress depending on plant species; however, it could have a synergistic impact when applied with salicylic acid. Similarly, there are many reports regarding the synergistic effect of salicylic acid with rhizobacteria; chemicals such as thymol, melatonin, chitosan, and hydrogen peroxide; and physical elements, including heat, on plant growth and on alleviating abiotic stresses [33,34,35,36,37,38].

3.2. Reduction in Lipid Peroxidation and Enhancement of Antioxidant Enzyme Activity with KJ40 and SA Combination

Water-deficit stress causes oxidative stress in plants due to the formation of reactive oxygen species (ROS), leading to lipid peroxidation, which is measured by an increase in malondialdehyde (MDA) [39]. To quantitatively determine stress alleviation in plants, lipid peroxidation by MDA was measured in unstressed plants and plants subjected to water-deficit stress. Plant MDA significantly increased after water-deficit stress (df = 2, F = 32.5414, p < 0.0001) (Figure 2). The MDA content did not show a significant difference between the control nor the treatments, including SA, KJ40, and KSA under unstressed conditions. However, it exhibited a marked decrease in single SA (p < 0.01), KJ40 (p < 0.05), and KSA combination (p < 0.05) compared to the control (Figure 2). These results indicate that both single and combined treatments specifically reduced plant lipid peroxidation under water-deficit stress. Water-stressed plants generally accumulate osmotolerant compounds such as proline, trehalose, and starch. These compounds aid in maintaining cellular hydration and stability under adverse conditions [40]; therefore, proline content was measured to determine the accumulation of plant osmotolerants. Water-deficit stress induced a significant accumulation of proline in napa cabbage plants compared to well-watered, unstressed conditions (df = 2, F = 61.1282, p < 0.0001) (Figure 2). Similar to MDA content under unstressed conditions, proline did not accumulate differently in the treated plants compared to control plants. However, under water-deficit conditions, single KJ40-treated plants exhibited a significantly lower proline accumulation compared to control-treated plants. These results suggest that both the single and combined treatments played a role in alleviating plant damage caused by water-deficit stress, and this effect was not driven by proline accumulation. It appears that the plants experienced less stress, as evidenced by the reduced need for proline accumulation. Similar observations of reduced proline accumulation under water-deficit conditions have been reported in a previous study on the application of the KJ40 strain to pepper plants against drought stress [22].
Meanwhile, plants protect themselves from deleterious singlet oxygen, free radicals, and hydrogen peroxide caused by abiotic stress through inducing antioxidant systems, such as superoxide dismutase (SOD) and catalase (CAT) [41]. In napa cabbage, when subjected to water-deficit stress, SOD (df = 2, F = 14.58072, p = 0.00027) and CAT (df = 2, F = 5.43850, p = 0.02228) activities were significantly influenced (Figure 3). The difference among treatments in both SOD and CAT was only shown under water-deficit conditions; in the case of plant SOD activity, only combining KSA significantly increased the activity, while in plant CAT activity, single SA and the KSA combination remarkably (p = 0.01) up-regulated the activity compared to the control, whereas the single KJ40 did not show a significant increase compared to the control (Figure 3). In our previous study regarding KJ40′s application to pepper plants under drought conditions [22], the KJ40-treated pepper plants also significantly reduced CAT and SOD activities compared to the control, which is consistent with the observations in this study on napa cabbages. Interestingly, when the KJ40 strain was applied with SA, the KSA combination-treated napa cabbage showed high SOD activity compared to the control, whereas the single SA treatment did not exhibit this effect. These results suggest the synergistic effect of the KSA combination on up-regulating antioxidant activity caused by water-deficit stress for scavenging ROS. It is well known that by converting highly reactive superoxide radicals into less harmful molecules, antioxidant enzymes help prevent the formation of more damaging ROSs and maintain a redox balance within plant cells. SOD is another key antioxidant enzyme that acts as the first line of defense against oxidative stress by catalyzing the dismutation of superoxide radicals (O2−) into oxygen (O2) and hydrogen peroxide (H2O2). The CAT is an essential antioxidant enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2), helping to prevent the accumulation of this ROS and subsequent oxidative damage to cellular components, such as proteins, lipids, and DNA [41]. Salicylic acid seems to play a role in enhancing antioxidant activity, including CAT, to cope with abiotic stress in plants. Cha et al. [42] revealed that CAT activity of exogenous SA-treated cabbage was higher than untreated Chinese cabbage seedlings from three to six days after drought treatment; Choudhary et al. [43] also demonstrated that salicylic acid enhances antioxidant activity in the presence of heat stress. However, Melo et al. [44] revealed that the 0.5 mM of salicylic acid treatment modulated increases in SOD and decreases in CAT under drought stress, suggesting that the antioxidant enhancement with salicylic acid could depend on plant species and genotypes, stress intensity, and other tested conditions. Meanwhile, the KSA combination could have a complementary or synergistic effect on enhancing antioxidant activity in water-deficit napa cabbage; each single treatment, SA and KJ40, significantly presented opposite statuses in CAT, whereas the combination treatment showed an enhancement in CAT and SOD activity compared to each single treatment. These results could be due to the expression of the SA effect overwhelming KJ40. In further studies, we will investigate the changes in the biostimulant activity of KJ40 by exogenously applying SA.

3.3. Single Salicylic Acid and Combination Treatments Induce Changes in Salicylic Acid Contents, Excluding Glucosinolate, in Napa Cabbage

To elucidate the impact of exogenous SA or combined treatments on napa cabbage’s endogenous content, the plant’s salicylic acid and glucosinolate contents were measured in single SA, KJ40, and KSA combination as well as distilled water-treated plants at 1, 3, and 5 days after stress (DAS). Salicylic acid content in napa cabbage was not influenced after water-deficit stress (df = 2, F = 2.12656, p = 0.14917); however, glucosinolate had increased significantly (df = 2, F = 1517.20156, p < 0.0001). Among treatments, single SA and the KSA combination induced salicylic acid contents that were higher than the control at 5 DAS. However, these differences were not present in unstressed plants (Figure 4). In plant glucosinolate contents, only single KJ40 had a negative impact on unstressed plants transiently; however, all treatments did not exhibit significance compared to the control at 1, 3, and 5 DAS under water-deficit stress conditions (Figure 4).
Salicylic acid in plants plays a multifaceted role in the acquisition of abiotic stress tolerance by regulating seed germination, root growth, stomatal closure; inducing osmotic adjustment; activating antioxidant defense mechanisms; modulating gene expression; and facilitating crosstalk with other hormone pathways [45,46,47]. Glucosinolates, which are rich in the Brassicaceae family, such as cabbage, broccoli, and mustard, are a group of sulfur-containing secondary metabolites [48]. These compounds accumulate to defend against pathogens and abiotic stresses. However, the accumulation and effects are contradictory depending on the duration and intensity of drought stress and plant species [45,49]. Glucosinolate is affected by other phytohormones, including methyl jasmonate, salicylic acid, and abscisic acid. Moreover, the application of exogenous salicylic acid induces the accumulation of glucosinolate contents in plants [50,51]. However, the effect of salicylic acid on glucosinolate content is less obvious [52]. In this study (Figure 4), water-deficit stress did not induce the accumulation of salicylic acid in napa cabbages over time; however, the exogenous application of SA, including single SA and the KSA combination, increased endogenous SA contents. The increasing endogenous salicylic content could impact the plant’s defense system against water-deficit stress. On the other hand, glucosinolate contents were significantly affected by water-deficit stress, but they were not differently accumulated in the treatments. These results suggest that the application of the single SA or the KSA combination treatments may not be involved in the glucosinolate-dependent tolerance to water-deficit stress. Regarding the results of napa cabbage’s fresh weight under water-deficit conditions in Figure 1, the KSA combination could be complementary; SA enhances tolerance to stress, while KJ40 redeems potential growth limitations via the accumulation of endogenous salicylic acid.

3.4. Impact on Soil Microbial Profiles and Activity

Based on community-level physiological profiling (CLPP) by EcoPlates™ containing 31 organic carbon substrates, soil metabolic potential was assayed in Figure 5. The average well color development (AWCD), which indicates the general potential metabolic activity of the microbial community, was not significantly affected by water-deficit stress (df = 2, F = 1.38389, p = 0.243315) at 5 DAS (Figure 5a). Only the KSA combination statistically decreased in AWCD compared to single KJ40 treatment under the water-deficit condition (Figure 5a). However, substrate average well color development (SAWCD), including amine, amino acids, carbohydrates, carboxylic acids, phenolic compounds, and polymers, was changed by treatments, such as single SA, KJ40, and the KSA combination compared to the control (Figure 5b). Under unstressed conditions, when the single treatments were applied, soil metabolic activity related to amines was increased compared to the control (Figure 5b). Under water-deficit stress conditions, the treatments had different impacts on SAWCD compared to the control. Specifically, in the case of the KSA combination, there was a drastic decrease in AWCD, except carbohydrate, including amino acids, carbohydrates, polymers, amines, phenolic compounds. These phenomena contrasted the effects that single SA and KJ40 displayed (Figure 5b). Osmotic-stressed plants generally rely on the synthesis and accumulation of soluble proteins, sugars, sugar alcohols, amino acids, and ammonium compounds, such as osmoprotectants or osmolytes [53]. Plants release a large variety of compounds into the rhizosphere as root exudates, including carbohydrates, amino acids, and organic acids, and phenolic compounds, and their composition could be changed by various stresses, such as drought, leading to a negative impact on soil microorganisms [54]. Water deficit induces a modification of amino acid metabolism and exudation in pea plants [55]; this stress can influence the translocation of assimilated nitrogen from the shoots to the roots, as well as the composition of exudation of amino acids. Although the impact of the changes on soil water retention is still greater, this could have an impact on rhizosphere microorganisms, which obtain nutrients from root exudates. As a result of Figure 5c, soil metabolic microbial activity responded differently depending on the chosen treatment against water-deficit stress. Representatively, the changes in AWCD regarding carboxylic acid, phenolic compounds, and polymers in KJ40-treated napa cabbages were greater under water-deficit stress (Figure 5c). On the other hand, the amounts of amino acids, amines, carboxylic acid, phenolic compounds, and polymers were lower in plants treated with the KSA combination under water-deficit conditions. These situations could be due to the KSA combination having a significant alleviating effect on water-deficit stress in napa cabbages, and it is involved in the decrease in the stress-related release of osmotolerants and root exudation. As demonstrated in Figure 1 and Figure 2, the KSA combination only showed a growth increase under water-deficit conditions and a decrease in lipid peroxidation without proline accumulation. Therefore, in this study, the KSA combination could have a distinct mechanism for alleviating water-deficit stress in napa cabbages that is different from the single SA and KJ40 treatments; it has a potential relation to the arrangement of the metabolic composition of the plants. It will need to be understood whether less-stressed plants subjected to treatments express changes in root exudates and soil metabolic microbial activity or if the treatment causes changes in the soil microbiome, resulting in a decrease in plant damage by water-deficit stress.
For the investigation of microbial activity in soil, beta-glucosidase, which is involved in the degradation of cellulose as a carbon source in soils, and FDase, which represents total microbial metabolic activity, were assayed. In Figure 6, regarding water-deficit stress’s impact on beta-glucosidase (df = 2, F = 121.08423, p < 0.0001) and FDase activity (df = 2, F = 8.7580, p = 0.004), soil beta-glucosidase under the water-deficit condition was higher than that of unstressed condition; moreover, the single SA treatment significantly increased beta-glucosidase activity compared to the control (Figure 6). On the other hand, total microbial activity when using the FDase assay was lower when the plants were under water-deficit stress, but the difference between the treatments was not evident (Figure 6). According to the results of Tomar et al. [56], soil enzymes, except beta-glucosidase, were significantly correlated with soil moisture. Additionally, soil moisture and temperature are important factors for determining soil microbiological processes, including carbon cycling, which is correlated with enzyme activity. On the other hand, beta-glucosidase activity was influenced by water-deficit stress in this study, and single SA also increased the enzymatic activity of the plants compared to the control (Figure 6). It could be elucidated that the plant could regulate root exudates and recruit certain microorganisms, which metabolize cellulose to glucose, which is sequentially used as a nutrient source for various microorganisms in order to protect them during water-deficit conditions. Soil microorganisms can help plants survive against drought stress with the release of amino acid osmolytes and compatible solutes, which are used for maintaining water and protein structure integrity, and they are used for scavenging ROS [57]. Further research needs to be carried out to understand the underlying mechanisms of these phenomena, including plant metabolic changes, such as root exudate components, soil microbial activity, and microbiome dynamics.

3.5. Impact of Salicylic Acid on the Population Dynamics of KJ40

To determine whether salicylic acid affects KJ40 strain’s survival, bacterial growth was measured with and without 0.5 mM of salicylic acid in TSB (Figure 7). Bacterial growth was significantly decreased in the TSB mixed with 0.5 mM salicylic acid as KSA compared to that without salicylic acid at 30 and 36 h after incubation (Figure 7). Meanwhile, in the rhizosphere soil samples of napa cabbages, 0.5 mM of salicylic acid transiently caused a decrease in the KJ40 population compared to the control at 4 days after treatment (DAT); however, it did not influence the KJ40 population at 7 DAT. When 1.0 mM of salicylic acid was added to KJ40, it significantly provoked a population reduction in KJ40 at 4 and 7 DAT. Regarding the results displayed in Figure 1, these results explain that less than 1 mM of salicylic acid did not impact the KJ40 population in the soil samples, making it seem like it acts a buffer against salicylic acid and shelters the strain, allowing KJ40 to coexist in the concentration of salicylic acid.

4. Conclusions

Overall, this study suggests that the combination of KJ40 and SA can have a synergistic effect on improving the growth and stress tolerance of napa cabbage, potentially by influencing plant hormones and soil microbial activity. The combination of KJ40 with SA, particularly at concentrations of 0.5 mM and 1 mM, showed synergistic effects in promoting napa cabbage growth under water-deficit conditions. While single applications of KJ40 or SA alone did not significantly enhance plant growth, their combination proved effective in mitigating the negative impacts of water deficit. The combined treatment reduced lipid peroxidation, which was evidenced by decreased MDA levels, under water-deficit stress, and enhanced antioxidant enzyme activities, such as SOD and CAT, which are crucial for scavenging ROS and maintaining a redox balance. Applying a combination of KJ40 and SA altered soil metabolic activity and substrate utilization patterns, especially affecting amino acids, amines, and phenolic compounds, suggesting that this caused potential changes in rhizosphere dynamics and nutrient cycling. However, soil microbial activities, including ß-glucosidase and FDase, were not significant. Salicylic acid concentrations below 1 mM did not significantly affect the KJ40 population in soil, indicating the strain’s resilience to salicylic acid exposure. On the other hand, higher SA concentrations led to a population reduction, suggesting a concentration-dependent effect. In summary, the combined application of SA and KJ40 holds promise for enhancing plant growth, mitigating oxidative stress, modulating soil metabolic profiles, and potentially improving plant resilience to water-deficit conditions. In future studies, it will be essential to understand these mechanisms by incorporating transcriptomics and microbiome analysis, elucidating root exudates, and by optimizing application strategies for practical agricultural use.

Author Contributions

Conceptualization, S.T.K. and M.K.S.; methodology, S.T.K.; software, S.T.K. and M.K.S.; validation, S.T.K. and M.K.S.; formal analysis, S.T.K. and M.K.S.; investigation, S.T.K.; resources, M.K.S.; data curation, M.K.S.; writing—original draft preparation, S.T.K.; writing—review and editing, M.K.S.; visualization, S.T.K.; supervision, M.K.S.; project administration, M.K.S.; funding acquisition, M.K.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Institute of Agricultural Sciences (project no. PJ01587101 (RS-2021-RD009876)) of the Rural Development Administration, Republic of Korea.

Data Availability Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest for this submission.

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Figure 1. Picture of unstressed and water-deficit stressed napa cabbage plants (A) and shoot fresh weights (B). The plants were treated with KJ40 and various concentrations (0, 0.1, 0.5, 1 mM) of salicylic acid as well as in combination treatments under unstressed (left column) and water-deficit stressed (right column) conditions. Asterisks indicate statistical differences between treatments using t-test (*, p < 0.05; **, p < 0.01).
Figure 1. Picture of unstressed and water-deficit stressed napa cabbage plants (A) and shoot fresh weights (B). The plants were treated with KJ40 and various concentrations (0, 0.1, 0.5, 1 mM) of salicylic acid as well as in combination treatments under unstressed (left column) and water-deficit stressed (right column) conditions. Asterisks indicate statistical differences between treatments using t-test (*, p < 0.05; **, p < 0.01).
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Figure 2. Malondialdehyde and proline contents of cabbage plants treated with single KJ40 and SA, and combination treatment (KSA) under unstressed condition (left column) and water-deficit stress (right column). Asterisks indicate statistical difference between treatments by t-test (*, p < 0.05; **, p < 0.01).
Figure 2. Malondialdehyde and proline contents of cabbage plants treated with single KJ40 and SA, and combination treatment (KSA) under unstressed condition (left column) and water-deficit stress (right column). Asterisks indicate statistical difference between treatments by t-test (*, p < 0.05; **, p < 0.01).
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Figure 3. Antioxidant enzyme activity, including superoxide dismutase and catalase, of cabbage plants treated with single KJ40 and SA, and KSA combination under unstressed condition (left column) and water-deficit stress (right column). Asterisks indicate statistical differences between treatments using t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
Figure 3. Antioxidant enzyme activity, including superoxide dismutase and catalase, of cabbage plants treated with single KJ40 and SA, and KSA combination under unstressed condition (left column) and water-deficit stress (right column). Asterisks indicate statistical differences between treatments using t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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Figure 4. Salicylic acid and glucosinolate contents of cabbage plants treated with single KJ40 and SA, and KSA combination under unstressed condition (left column) or water-deficit stress (right column). Asterisks indicate statistical differences between treatments using t-test (*, p < 0.05; **, p < 0.01).
Figure 4. Salicylic acid and glucosinolate contents of cabbage plants treated with single KJ40 and SA, and KSA combination under unstressed condition (left column) or water-deficit stress (right column). Asterisks indicate statistical differences between treatments using t-test (*, p < 0.05; **, p < 0.01).
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Figure 5. The average well color development (AWCD) index of soil samples at 72 h after incubation (A); heatmap of substrate average well color development (SAWCD) related to carbohydrates, amino acids, carboxylic acids, polymers, amines, and phenolic compounds (B) in rhizosphere soils treated with control (a), SA (b), KJ40 (c), and KSA (d); % changes of AWCD by stress treatment at 72 h after incubation (C). Asterisks indicate statistical differences between treatments using t-test (**, p < 0.01).
Figure 5. The average well color development (AWCD) index of soil samples at 72 h after incubation (A); heatmap of substrate average well color development (SAWCD) related to carbohydrates, amino acids, carboxylic acids, polymers, amines, and phenolic compounds (B) in rhizosphere soils treated with control (a), SA (b), KJ40 (c), and KSA (d); % changes of AWCD by stress treatment at 72 h after incubation (C). Asterisks indicate statistical differences between treatments using t-test (**, p < 0.01).
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Figure 6. Soil enzyme activity, including beta-glucosidase, and FDase assay of napa cabbage plants treated with single KJ40 and SA, and KSA combination under unstressed condition (left column) or water-deficit stress (right column). An asterisk indicates statistical differences between treatments using t-test (*, p < 0.05).
Figure 6. Soil enzyme activity, including beta-glucosidase, and FDase assay of napa cabbage plants treated with single KJ40 and SA, and KSA combination under unstressed condition (left column) or water-deficit stress (right column). An asterisk indicates statistical differences between treatments using t-test (*, p < 0.05).
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Figure 7. Population of KJ40 in tryptic soy broth with and without 0.5 mM of salicylic acid (left), and colonization at 1, 4, and 7 days after treatment (DAT). Asterisks indicate statistical differences between treatments using t-test (*, p < 0.05; ***, p < 0.001, ****, p < 0.0001).
Figure 7. Population of KJ40 in tryptic soy broth with and without 0.5 mM of salicylic acid (left), and colonization at 1, 4, and 7 days after treatment (DAT). Asterisks indicate statistical differences between treatments using t-test (*, p < 0.05; ***, p < 0.001, ****, p < 0.0001).
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Kim, S.T.; Sang, M.K. Synergistic Effects of Salicylic Acid and Bacillus butanolivorans KJ40 for Enhancing Napa Cabbage (Brassica napa subsp. pekinensis) Resilience to Water-Deficit Stress. Horticulturae 2024, 10, 618. https://doi.org/10.3390/horticulturae10060618

AMA Style

Kim ST, Sang MK. Synergistic Effects of Salicylic Acid and Bacillus butanolivorans KJ40 for Enhancing Napa Cabbage (Brassica napa subsp. pekinensis) Resilience to Water-Deficit Stress. Horticulturae. 2024; 10(6):618. https://doi.org/10.3390/horticulturae10060618

Chicago/Turabian Style

Kim, Sang Tae, and Mee Kyung Sang. 2024. "Synergistic Effects of Salicylic Acid and Bacillus butanolivorans KJ40 for Enhancing Napa Cabbage (Brassica napa subsp. pekinensis) Resilience to Water-Deficit Stress" Horticulturae 10, no. 6: 618. https://doi.org/10.3390/horticulturae10060618

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