Comparative Evaluation of Paclobutrazol and Rhizobacterium Variovorax sp. YNA59 Effects on Mitigating Drought Stress in Chinese Cabbage (Brassica rapa ssp. Pekinensis)

Abstract

Drought has severely impacted worldwide agricultural land, resulting in substantial yield loss and abiotic pressures in crops such as Chinese cabbage. Paclobutrazol (PBZ) and plant growth-stimulating bacteria have been extensively recommended to reduce a variety of stresses in crops. Considering these facts, we used PBZ and rhizobacterium Variovorax sp. YNA59, a microbe with potential plant growth-stimulating qualities, in Chinese cabbage under drought stress and non-stressed conditions, and we observed their impacts on morphological and physiological attributes. Our findings revealed a considerable improvement in total plant biomass growth after treatment with YNA59 (28%) and PBZ (8%) under drought conditions. In contrast, negative results of PBZ were observed under normal conditions, suppressing plant growth and reducing plant biomass. The increase in physiological parameters, such as chlorophyll content, relative water content, and photochemical efficiencies of PS II, were observed in YNA59-treated plants, followed by PBZ, especially under drought. Furthermore, drought stress significantly increased the endogenous phytohormone abscisic acid level, whereas PBZ and YNA59 inoculations significantly decreased it. Antioxidant analysis revealed that PBZ and YNA increased glutathione and catalase and decreased polyphenol oxidase and peroxidase levels. These findings suggested that rhizobacterium YNA59 could be more significant for conferring water stress in the Chinese cabbage plant than PBZ.

1. Introduction

Drought is a major abiotic barrier to crop productivity that has had a considerable impact on worldwide crop production in recent decades [1,2]. In the past four decades, the percentage of lands affected by drought, based on the data of water availability, agricultural production, food security, and rural livelihood, has increased, with a greater impact on farmers than any other natural hazard (FAO, 2021) (Appendix A). By the first half of the twenty-first century, water drought episodes are expected to rapidly deteriorate and reach record heights unseen in human history [3]. Additionally, the population of the globe is fast expanding and is projected to reach nine billion people by the year 2050 [4]. In these situations, reducing drought stress in crops is essential to address the likelihood of food insecurity [5]. Conferring stress resistance in crops through external supplements is one of the fundamental scientific methods; hence, it is crucial to understand the underlying physiological response of particular crops. In general, plant metabolism is harmed by drought conditions, which disturb plant–water relations and reduce leaf size, stem extension, and root proliferation, resulting in ionic imbalance, oxidative damage, and a decrease in gas exchange rates, all of which ultimately cause plant mortality [6,7]. Hence, in response to drought stress, plants activate the antioxidant system and modulate endogenous phytohormones, especially the ABA channel, which in turn activates guard cells to control stomatal conductance and maintain high moisture levels by regulating the water potential of the leaf and the soil [8,9].

It has been demonstrated that plant growth-promoting microorganisms (PGPMs) serve a functional role in the interaction of ions in plants while also improving the ability of soils and plants to hold water during drought stress [10]. Rhizobacteria functions through symbiotic associations with plant roots, regulating different mechanisms of plants, such as stimulating phytohormonal activity and increasing root surface area, siderophore production, mineral solubilization, and various chemical signals associated with plant roots [11,12]. These phenomena have been reported in several biotic and abiotic stresses in varieties of crops, such as salt in rice [13,14], drought in potato [15], heat stress in maize and wheat [16,17], heavy metal toxicity in tomato [18], and others. Additionally, agrochemicals are being replaced to a large extent by PGPMs in the production of sustainable food [19].

Similarly, a triazole plant growth regulator known as paclobutrazol (PBZ) [(2RS, 3RS)-1-(4-chlorophenyl)-4, 4-dimethyl-2-(1, 2, 4-triazol)-pentan-3-ol] is used to protect plants from a variety of abiotic challenges, including salt, flooding, and water deficiency stress [20,21]. Numerous advantages of PBZ treatment have been extensively documented, including increased crop output, increased plant stress tolerance, improved fruit and grain quality, improved plant–water relations, and increased membrane stability index [22,23].

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a major vegetable crop in Korea, since it is used to make Kimchi, one of the country’s most popular meals. Kimchi is filled with a variety of spices, such as garlic, ginger, and scorching red pepper [24]. Chinese cabbage is widely cultivated in Asia, especially in Korea, Japan, and China, due to its agricultural and economic value [25,26]. It is highly susceptible to extreme and atypical weather [27,28]. To confer stress tolerance in Chinese cabbage, the practice of bio-stimulants treatment could result in significant improvement in its growth and internal metabolism [25].

Despite the significance of PGPM and PBZ, there are certain limitations in their application. For example, PBZ has also been reported for threat, due to its high stability and potential for bioaccumulation [29], resulting toxicity and death in zebra fish [30], soil pollution, and inhibited growth in potato [31]. In contrast, the microbial activity might be highly unstable, due to the fluctuation in environmental conditions, as compared to PBZ. Considering these facts, we tried to compare the efficiencies of rhizobacteria vs. PBZ in Chinese cabbage so that we could screen the effective bio-stimulant for future research and as a recommendation for an industrialization process. The current research lays out a basic idea on the appropriate selection of biological tools, especially for conferring the drought tolerance in Chinese cabbage.

2. Material and Methods

2.1. Plant Materials and Selection of Rhizobacterium and Paclobutrazol

We collected several reports of our crop physiology laboratory, based on the plant–microbe interaction, where we found the bacterial strain YNA59 to be one of the efficient strains in conferring drought stress resistance in crops. The innate ability of YNA59 to promote plant growth was previously reported in our experiment by Kim et al. [32], in which YNA59 displayed a number of features that were associated with stimulating plant development, including ABA and sugar-producing activities. Furthermore, the strain YNA59 exhibited high tolerance to oxidative stress, including hydrogen peroxide (H₂O₂), and the culture broth contained superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities [32]. Hence, as a continuation of our earlier experiment, YNA59 was used for further investigation. Additionally, paclobutrazol, (Kyungnong Co., 0.39% paclobutrazol, Seoul, Republic of Korea) and barrel-headed Chinese cabbage, Pekinensis group (napa cabbage), was used for the plant experiment.

2.2. Experimental Design

The experiment was conducted on two sets: (A) without stress (NS; NT, YNA59, and PBZ) and (B) with drought stress (DS; NT, YNA59, and PBZ).

2.3. Plant Experiment

The plant experiment was carried out in the Kyungpook National University’s green house in Daegu, Republic of Korea. The Luria–Bertani (LB) medium-containing bacterial culture of YNA59 was cultured for a week at 30 °C in a shaking incubator at 150 rpm. The bacterial solution of 200 mL (2.36 × 10⁸ cfu/mL) was centrifuged at 6000 rpm, and the pellets were separated. The obtained pellets were diluted with 200 mL of sterilized water for inoculation. The Chinese cabbage seeds were sown in a 64-holed plastic tray (27.94 × 53.34 × 4.5 cm) containing autoclaved horticultural substrate and grown under a greenhouse-conditions (26 ± 5 °C temperature) and 65 ± 10% RH. The formulation of the horticultural substrate was as follows: peat moss (10–15%), coco peat (45–50%), zeolite (6–8%), and perlite (35–40%), along with NH+ (≈0.09 mg·g⁻¹), NO₃ (≈0.205 mg·g⁻¹), PO (≈0.35 mg·g⁻¹), and KO (≈0.1 mg·g⁻¹); two-week-old Chinese cabbage (20 per treatment) was transplanted into the pot (33.0 × 27 × 33 cm³) and treated with the bacterial solution prepared from the pellets (20 mL day⁻¹) subsequently for five days. After a week, drought stress was generated by limiting the water supply to 50 mL pot⁻¹ week⁻¹, with enough water supply on the control (200 mL pot⁻¹ week⁻¹). Two times each week, the PBZ standard concentration (1.25 mL/Lit solution) was prepared, with a total of two treatments (once per week). Foliar treatment was performed until the leaves were sufficiently wetted. Two weeks after treatment, morphological parameters (root length, shoot length, fresh weight, leaf length, and leaf width) were measured, and plants were harvested in liquid nitrogen, freeze-dried, pulverized, and stored at −80 °C until analysis.

2.4. Analysis of Biochemical Properties

2.4.1. Chlorophyll Measurement

Leaf chlorophyll: Chlorophyll a (Chl a) and chlorophyll b (Chl b) were ascertained by the spectrophotometric analysis of chemically extracted pigments, as described by Arnon [33]. Briefly, one gram of freeze-dried, pulverized Chinese cabbage leaves was extracted in 100% ethanol at room temperature using a shaker and centrifugation. Pigment absorption was then measured spectrophotometrically at 663 and 645 nm (Thermo Fisher Scientific, Waltham, MA, USA). Chlorophyll fluorescence was measured using OS5p+ (Opti-Sciences Inc. 8 Winn Avenue, Hudson, OH, USA). The data were recorded everyday around 10–11 a.m. for seven days. The upper part of the 3rd leaf was clipped and left for a while, and the data were recorded.

2.4.2. Relative Water Content Measurement

The relative water content (RWC) of leaves was calculated using a method mentioned by Lubna, et al. [34]. The Chinese cabbage leaves were harvested, weighed (fresh weight; FW), and immersed in distilled water overnight. At the end, the turgid weight (TW) was measured. It was then kept in a preheated oven at 75 °C for 48 h, and the dry weight (DW) was measured. The RWC was finally determined using the following formula:

RWC % = ([FW − DW]/[TW − DW]) × 100.

2.4.3. Relative Water Loss (ELWL) Calculation

The ELWL content was also calculated according to the protocol described by Lubna et al. [34]. To determine the ELWL content, the cabbage leaves were harvested, and the fresh weight was measured, as mentioned in the RWC measurement section. The samples were then dried at 28 °C for 6 h (W6h), and the weight was determined. To determine the sample’s total dry weight, the sample was then dried at 70 °C for 24 h. Eventually, the following formula was used to determine the ELWL content:

ELWL (%) = (FW − W6h)/(FW − DW) × 100

2.4.4. ABA Analysis

The ABA content of Chinese cabbage shoots was extracted and quantified using the technique reported by Kim, et al. [35]. In brief, five grams of the freeze-dried sample were suspended with isopropanol and acetic acid (95:5 v/v) and then shaken for 3 h in a shaker at 120 rpm. The solvent obtained was filtered, and ABA internal standard [(±) −3,5,5,7,7,7-d⁶] was added, followed by washing with 1 N NaOH and the removal of chlorophyll by CH₂Cl₂. The solution was further passed through EtoAc and suspended in polyvinylpolypyrrolidone for one hour in a shaker at 120 rpm. Next, the solvent was filtered, evaporated, and extracted with ethyl acetate/diethyl ether. The ABA extracts were dried with N_2, methylation was performed with diazomethane, and the product was washed with CH₂Cl₂. The ABA was analyzed through injection on GC-MS/SIM (6890 N Network GC System and 5973 Network Mass Selective Detector: Agilent Technologies, Santa Clara, CA, USA).

2.4.5. Antioxidant Activities

• Glutathione (GSH) Measurement

GSH was quantified according to the protocol described by Khan, et al. [36]. In brief, the freshly harvested leaf after storage at −80 °C for a few hours was homogenized with liquid nitrogen using a mortar and pestle and extracted with 80% (v/v) trichloroacetic acid. The extract was then centrifuged at 1000 rpm for 15 min at −4 °C; the supernatant was collected and afterwards, suspended in 15f0 mM NaH₂PO₄ (pH 7.4) containing 0.5 mL of Ellman’s reagent for 10 min and kept in the dark. The absorbance was determined at 412 nm and calculated using a standard curve.

• Catalase (CAT) Measurement

Catalase was determined according to the method described by Aebi [37]. Frozen samples were extracted with a solution (50 mM Tris HCl, pH 7.0 3 mM MgCl₂, 1 mM EDTA, and 1% PVP), and the supernatant was separated and mixed with the solution (0.1 M Phosphate buffer pH 7.0, 0.2 M H₂O₂). The degree of decomposition of H₂O₂ was measured at 240 nm using a spectrophotometer (Multiskan GO; Thermo Fischer Scientific, Vantaa, Finland).

• Polyphenol oxidase (PPO) measurement

PPO was calculated according to the method described by Khan et al. [38]. In brief, the samples were extracted using extraction buffer (50 mM sodium phosphate (pH 7.0), 1% polyvinylpolypyrrolidone (PVP; w/v), and 0.1 mM EDTA) and centrifuged at 12,000 rpm at 4 °C for 15 min. The absorbance was taken at 420 nm using a spectrophotometer (Multiskan GO; Thermo Fischer Scientific, Vantaa, Finland)

• Peroxidase (POD) measurement

To determine the POD, the fresh sample was extracted using 0.1 M phosphate buffer (pH 6.8) and centrifuged at 12,000 rpm at 4 °C for 15 min. Next, the steps described by Pütter [39] were used to calculate the POD content in Chinese cabbage shoots. In brief, the supernatant obtained was mixed with 0.1 M phosphate buffer, 50 μM pyrogallol, and 50 μM H₂O₂ and left to sit at room temperature for 5 min. Subsequently, 5% of H₂SO₄ was mixed and measured at 420 nm using a spectrophotometer (Multiskan GO; Thermo Fischer Scientific, Vantaa, Finland).

2.5. Statistical Analysis

The present study was conducted in a completely randomized design (CRD), and the data were statistically analyzed with SAS version 9.4 software (SAS Institute, Cary, NC, USA). The mean values among treatments were compared using Duncan’s multiple range test (DMRT) at p ≤ 0.05.

3. Results

3.1. Morphological Characteristics

The morphological appearance of plants aids in visually determining the level of stress in plants, from which we can create a specific approach to bestow tolerance in crops. Our experiment results showed that the physical attributes of plants, such as shoot length, root length, total fresh weight (root + shoot), leaf length, and leaf width were significantly decreased by PBZ treatment by 31%, 5%, 11%, 32%, and 17%, respectively, when compared to NT. However, the YNA59 treatment considerably improved these attributes under normal condition.

Under drought stress, YNA59 inoculation increased the shoot length, root length, fresh weight, leaf length, and leaf width by 25%, 30%, 28%, 23%, and 24%, respectively, when compared to NT. Contrastingly, PBZ treatment reduced the shoot length, root length, leaf length, and leaf width by 11%, 13%, 29%, and 9% while increasing the fresh weight by 8%, when compared to NT. Furthermore, upon visual observance, it was seen that the PBZ application reduced the plant growth, especially under normal conditions, whereas YNA59 improved the growth under both normal and stressed conditions, as shown in Figure 1 and Table 1.

3.2. Chlorophyll Measurement

The YNA59 application showed a minor increase in Chl a content, whereas PBZ treatment significantly enhanced the Chl a by 58% and 26% in NS and DS, respectively, when compared to the control. A similar trend was observed in Chl b, where YNA59 application showed a slight increase in Chl b, whereas PBZ treatment significantly increased the Chl b by 83% and 80% in NS and DS, respectively. Chlorophyll fluorescence was increased by YNA59 and PBZ by 10% and 8%, respectively, under DS. No significant differences were observed under NS, as shown in Figure 2.

3.3. Relative Water Content Measurement

Relative water content is a significant parameter to determine the osmotic potential of the plant and the level of stress. Here, the drought stress significantly reduced the RWC by 47% in the Chinese cabbage plant when compared to the control. However, YNA59 inoculation and PBZ treatment considerably increased the RWC content by 54% and 38%, respectively, under DS. Only minor differences were observed under non-stressed conditions upon treatment with YNA59 and PBZ when compared to control, as shown in Figure 3A.

3.4. Relative Water Loss (ELWL) Measurement

Relative water loss is another major parameter to check the moisture percentage of plant shoots. Here, drought stress decreased ELWL content by 30%, which was limited to 20% and 25% for YNA59 and PBZ treatments. Moreover, under normal conditions, YNA59 and PBZ treatments resulted in losses of 19% and 7%, respectively, as shown in Figure 3B.

3.5. Quantification of Endogenous Phytohormone Abscisic Acid (ABA)

Abscisic acid is a significant growth regulator that responds swiftly to plant abiotic stress, particularly drought stress, via cross-signaling. The level of ABA varies according to the water uptake in plants, moisture level, and transpiration rate. In our study, we observed the significant elevation of ABA (1868 ng g⁻¹ D.W) in Chinese cabbage shoots without treatment under drought when compared to the control (225 ng g⁻¹ D.W). The ABA content was greatly reduced with YNA59 (1683 ng g⁻¹ D.W) and PBZ (1667 ng g⁻¹ D.W) applications when compared to control under drought (1868 ng g⁻¹ D.W). Under normal conditions, ABA concentrations were limited to 225, 292, and 428 ng g⁻¹ D.W in control, YNA59, and PBZ, respectively, as shown in Figure 4.

3.6. Analysis of Antioxidant Enzymes

Antioxidant enzymes such as glutathione (GSH) and catalase neutralize the harmful oxygen radicals in plants and protect the plants from cell injury. Our experiment results showed that YNA59 and PBZ treatments significantly increased the GSH content by 121% and 78%, respectively, under drought. Under normal conditions, the YNA59 application dropped the GSH content by 36%, whereas the PBZ treatment enhanced the GSH content by 118% when compared to the control as shown in Figure 5A. A similar trend was observed in the case of CAT, where the YNA59 and PBZ increased CAT by 5% and 22%, respectively, under drought and 12% and 4% under normal conditions as shown in Figure 5B.

3.7. Measurement of Polyphenol Oxidase (PPO) and Peroxidase (POD)

PPO and POD are the major antioxidants that are involved in various cross-signaling with other phytochemicals and regulating plant metabolism. In our results, we observed that the PPO content was significantly increased by 50% by drought, which was gradually decreased by 34% and 31% by YNA59 and PBZ treatments when compared to the control. Similarly, POD content was decreased by 22% by YNA59 and 11% by PBZ treatment under drought. Under normal conditions, both PPO and POD showed a non-significant difference in YNA59 and PBZ treatments when compared to the control Figure 6A,B.

4. Discussion

The impacts of environmental stressors, chemical pollution, climatic change, and economic inflation on agriculture must be immediately countered [40,41]. The experts are currently working feverishly to develop a method for minimizing plant stresses for sustainable agriculture. Despite attempts, there has been minimal success in mitigating stress, such as drought, due to the challenging issue of climate change and the rapidly escalating population. Thus, the primary purpose of the current study was to improve drought stress by improving the physiological context of the Chinese cabbage plant. Paclobutrazol and plant growth-promoting rhizobacteria are considered efficient bio-stimulants to combat several biotic and abiotic stresses [42,43]. This study clarified the relative effects of PBZ and YNA59 in improving Chinese cabbage growth and internal metabolism.

Drought stress causes ionic disturbances in plants, causing osmotic stress, which may ultimately lead to cell necrosis. The results of the current study showed that the plant’s physical growth was significantly decreased with drought stress. The PBZ treatment suppressed the plant growth under normal conditions; however, it improved these characteristics under drought stress. However, the rhizobacterium YNA59 treatment enhanced plant vegetative growth under both normal and drought-stressed conditions. From these results, we can infer that PBZ application may trigger negative results during normal conditions. Hence, determining the appropriate dose of PBZ is crucial. The beneficial aspect of PBZ as a positive growth regulator under drought has been demonstrated by several authors, such as Sheikh et al. [44] in rye grass and Davari et al. [45] in safflower. Additionally, Still et al. [46] demonstrated that the growth retardant by PBZ in tomato seedlings was associated with high stress tolerance. From these results, we can infer that PBZ application may trigger both positive and negative results. Hence, determining the appropriate dose of PBZ is crucial for optimum plant growth. Furthermore, our results agreed with our previous experiment by Yuna et al. [32], where the beneficial association of YNA59 and plant rhizosphere improved plant growth through regulating plant growth regulators, which confer stress tolerance in crops. Our results are further supported by several authors, such as Batool et al. [15], Ferioun et al. [47], and Gul et al. [48], who reported that rhizobacterium inoculation improved the growth of crops under drought stress through improving physiological attributes, such as phytohormone modulation and antioxidant activation.

Crops’ ability to withstand stress is significantly influenced by the cross-signaling of phytohormones and antioxidants [49]. Endogenous phytohormones such as ABA are important for modulating stomatal conductance and initiating an adaptive response to drought stress [50]. In the current investigation, endogenous ABA content greatly rose in non-treated plants, which is a key and usual response to abiotic stress, but after PBZ treatment and YNA59 inoculation, the endogenous plant ABA content significantly decreased. The control of ABA in the current investigation was consistent with the earlier findings by Shahzad et al. [51] and Kang et al. [52].

Antioxidant systems in general counteract the oxidative stress generated by reactive oxygen species (ROS), which include PPO, POD, CAT, GSH, SOD, APX, and others [53,54]. Our study showed an increase in CAT and GSH, whereas there were decreases in PPO and POD upon treatment with YNA59 and PBZ under drought stress in Chinese cabbage plants.

Our results are in line with Parveen et al. [55], who reported an increase in malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents and antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)) in maize under drought. A similar trend was reported by Korrfler et al. [56] and Kocsy et al. [57], who reported an increase in GSH under osmotic stress in wheat, maize, and arabidopsis, respectively. An increase in CAT and a decrease in POD activity are related to less accumulation of the hydroxyl radical (OH) that decreases malondialdehyde in plant shoots, lowering lipid peroxidation and stimulating growth, as reported by Shi et al. [58] in the case of tomato. The phenomenon of catalase in detoxifying H₂O₂ is associated with converting O₂ formed by SOD through dissociating 2H₂O₂ to O₂ + 2H₂O [59]. Additionally, higher quantities of ABA were produced in response to glutathione administration [60], which is also connected with the management of leaf water content [61].

Plants under drought stress continuously lose water through transpiration and evaporation into the atmosphere, and their ability to absorb water is hampered by lower soil moisture levels. Since we observed a higher level of ABA under drought, these might be linked to causing higher stomatal conductance, resulting in an increased transpiration rate and ultimately retaining the lower moisture level in the plant. Another phenomenon might be the closing of the stomata, which restricts gas exchange between leaves and the atmosphere and lowers the CO₂ to O₂ ratio, which is one of the earliest reactions of plants to dryness [62,63]. ABA is inter-linked with regulating various transcriptomes, such as OsERF71 in rice, detoxifying the toxic radical under drought stress [64]. Moreover, an increment in proline triggered by ABA alleviates the adverse effect of water stress, as observed in wheat cultivars [65]. Our results are further supported by Shahzad et al. [51], who reported the application of ABA-producing endophytic bacteria on augmenting salinity stress in rice.

Moreover, the application of the rhizobacterium and PBZ improved plant morphological attributes, chlorophyll, and relative water content, reducing the relative water loss of Chinese cabbage under drought. Our results are in line with Yuan et al. [66], who reported that water stress caused a marked reduction in chlorophyll a, chlorophyll b, total chlorophyll fluorescence, and yield of tomato plants. High tolerance is linked to osmotic equilibrium, according to the available research. By controlling the ion transport system in plant tissues, plant adaptation and sensitivity are significantly influenced. PGPR normally prevents harmful ion inflow and preserves the osmotic balance in plants. Similar action is reported by the PBZ application. Moreover, the suppressed plant growth might be due to the higher oxidative stress exerted by drought. These phenomena are agreed upon by several authors, such as Hasanuzzaman et al. [67] and Davari et al. [45], who reported that the content of photosynthetic pigments and proline, RWC, cell membrane stability, enzymatic defense system, and dry matter accumulation were improved by the foliar application of PBZ under stress. In our experiment, we observed that the PBZ treatment improved the physiological phenomena of the plant at the cost of reduced height and length; however, the biomass increased. The physiological cause associated with this mechanism of paclobutrazol’s ability to reduce plant height may be due to its anti-gibberellin effects. According to the report, PBZ inhibits the gibberellins synthesis and accumulates more precursors in the terpenoid pathway, which may trigger the production of abscisic acid; hence, PBZ is also known as a growth retardant and a stress protectant [68].

5. Conclusions

To sum up, our study demonstrated that the application of YNA59 and PBZ regulated endogenous phytohormones, strengthened the antioxidant systems, and improved the physiological and morphological parameters of Chinese cabbage under drought stress. However, a significant decrease in plant fresh weight was observed by PBZ treatment under normal conditions, whereas YNA59 improved the overall physiological and physical attributes of Chinese cabbage plants. From the ecologically friendly aspect, with the high probability of PBZ inducing environmental pollution and aquatic hazards, YNA 59 could be more efficient when compared to PBZ treatment in mitigating water stress in the case of Chinese cabbage.

Author Contributions

Conceptualization and methodology, S.-M.K.; investigation, S.-M.K. and Y.-N.K.; writing—original draft preparation, A.A.; supervision, project administration, and funding acquisition, I.-J.L. and S.-M.K. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no roles in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

NT	No treatment
YNA59	Rhizobacterium Variovorax sp. Inoculum
PBZ	Paclobutrazol
NS	No stress
DS	Drought stress

Footnotes

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Figures and Table

Figure 1. Visual appearances of the morphological attributes of Chinese cabbage under normal and stressed conditions (NT, no treatment; YNA59, bacterial inoculum; PBZ, paclobutrazol).

Figure 2. (A,B) Chlorophyll a, chlorophyll b, and (C) chlorophyll fluorescence in Chinese cabbage following inoculation with PBZ and YNA59 under normal and drought-stressed conditions. Each data point represents the mean of at least eight replicates. Error bars represent standard deviations. Bars with different letters are significantly different from each other at p ≤ 0.05.

Figure 3. Effects of PBZ and YNA59 inoculation on relative water content (RWC; (A)) and relative water loss(ELWL; (B)) in Chinese cabbage. Each data point represents the mean of at least eight replicates. Error bars represent standard deviations. Bars with different letters are significantly different from each other at p ≤ 0.05.

Figure 4. The endogenous phytohormones abscisic acid (ABA) level of Chinese cabbage following inoculation with PBZ and YNA59 under normal and drought stress conditions. Error bars represent standard deviations. Each data point represents the mean of at least three replications. Bars with different letters are significantly different from each other at p ≤ 0.05.

Figure 5. Effects of PBZ and YNA59 treatments on antioxidant activities (A) GSH and (B) CAT. Error bars represent standard deviations. Each data point represents the mean of at least six replications. Bars with different letters are significantly different from each other at p ≤ 0.05. Catalase 1 unit refers to the amount that dissolves 1 µmole of H2O2 in 1 min at pH 7.0 and 25 °C.

Figure 6. Effect of PBZ and YNA59 treatment on antioxidant activity. (A) PPO and (B) POD. Error bars represent standard deviations. Each data point represents the mean of at least six replications. Bars with different letters are significantly different from each other at p ≤ 0.05. One unit of POD and PPO was defined as an increase of 0.1 units of absorbance.

Table 1

Effects of PBZ and YNA59 on morphological characteristics of Chinese cabbage.

	Shoot Length(cm)	Root Length(cm)	Total Fresh Biomass(g)	Leaf Length (cm)	Leaf Width (cm)
None Stress
NT	20.9 ± 1.22 a	15.2 ± 0.87 b	6.9 ± 0.31 b	19.4 ± 1.23 a	8.8 ± 0.74 a
YNA59	21.2 ± 1.53 a	18.4 ± 1.38 a	7.2 ± 0.27 a	20.7 ± 1.04 a	9.6 ± 0.56 a
PBZ	14.4 ± 0.98 b	14.4 ± 20.95 b	6.1 ± 0.12 c	13.0 ± 0.78 b	7.3 ± 0.33 b
Drought Stress
NT	15.1 ± 2.42 b	13.1 ± 0.65 b	5.0 ± 0.12 c	14.4 ± 0.78 b	6.7 ± 0.54 b
YNA59	18.8 ± 1.01 a	17.1 ± 1.33 a	6.4 ± 0.25 a	17.7 ± 0.69 a	8.3 ± 0.36 a
PBZ	13.4 ± 0.87 b	11.3 ± 0.93 a	5.4 ± 0.21 b	10.1 ± 0.56 c	6.1 ± 0.57 b

Each value represents the mean ± SD. Each data point represents the mean of at least eight replicates. Different letters in the column after the mean values represent the least significant differences at p ≤ 0.05.

Appendix A

Reference for FAO (2021). Drought and Agriculture. Available online at: https://www.fao.org/land-water/water/drought/droughtandag/en/ (accessed on 12 July 2023).

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