Phosphate-Solubilizing Enterobacter ludwigii AFFR02 and Bacillus megaterium Mj1212 Rescues Alfalfa’s Growth under Post-Drought Stress

: Drought stress is a prevalent environmental stress that adversely affects agricultural industries worldwide. In this study, bacterial isolates, AFFR02 and Mj1212, showed tolerance to polyethylene glycol-induced (PEG) drought stress (approximately 15%) and possess strong phosphate-solubilizing capacity. Moreover, we investigated the plant growth attributes, chlorophyll content, and ion uptake in alfalfa plants ( Medicago sativa L) inoculated with isolates AFFR02 and Mj1212 under drought stress. We observed that drought stress drastically affects alfalfa’s growth attributes: shoot length: SL (24.88%), root length: RL (29.62%), shoot fresh weight: SFW (49.62%), root fresh weight: RFW (45.09%), stalk diameter: SD (52.84%), and chlorophyll content: CC (19.2%). However, in bacterial-inoculated alfalfa plants, the growth attributes signiﬁcantly recovered were SL (12.42%), RL (21.30%), SFW (50.74%), RFW (46.42%), SD (76.72%), and CC (17.98%). In drought-stressed alfalfa plants, we observed a signiﬁcant decrease in the relative water content (7.45%), whereas there was an increase in electrical conductivity (68.87%) and abscisic acid contents (164.42%). Antioxidant analysis showed a signiﬁcant increase in total phenolic content (46.08%), DPPH-scavenging activity (39.66%), total ﬂavonoid (13.68%), and superoxide dismutase (28.51%) in alfalfa treated with drought stress and bacterial isolates AFFR02 and Mj1212 simultaneously. More-over, an increase in inductively coupled plasma (ICP) analysis of potassium (17.98%), phosphorous (11.14%), calcium (3.07%), and magnesium (6.71%) was recorded for bacteria-inoculated alfalfa plants under drought stress. In conclusion, bacterial isolates AFFR02 and Mj1212 enhance alfalfa growth under drought stress. Therefore, the isolates could be used as potential candidates in smart-climate agricultural practices in drought-stricken areas worldwide.


Background
Environmental factors, such as drought, salinity, temperature, heavy metals, and freezing stress, are important agricultural problems that impose stress on crop plants and cause widespread crop losses worldwide [1][2][3][4][5][6][7][8][9]. The continuous increase in temperature and water scarcity is increasing the frequency of severe drought conditions [10]. Drought stress is defined as the scarcity of water available to plants, and 50% of the world's arid or semiarid land is subjected to drought stress [11]. Drought stress intervenes in the normal biochemical, physiological, and morphological processes of plants by reducing leaf size,

Isolation, Screening, and Identification
Rhizospheric bacteria were isolated from Seosan, Chungcheongnam-do Province, and Geongbuk-do Province in the Republic of Korea, according to the detailed method of [53,54]. All isolates were screened for different PEG (0%, 5%, 10%, and 15%) concentrations and phosphate solubilization. To elevate the drought tolerance of isolated rhizospheric bacteria, different PEG (0%, 5%, 10%, and 15%) concentrations were added in Luria broth (LB) media and kept in a shaking incubator (120 rpm; 28 • C) for 4 day. The optical density (OD) (600 nm) was taken at a regular interval of 12 h for 4 d by using a spectrophotometer (Shimadzu, Kyoto, Japan). The National Botanical Institute Phosphate media (0.5%) was used to inoculate isolated microbes, and they were incubated at 30 • C [55,56]. The bacterial isolate formed a cleared halo on plates, indicating phosphate solubilization. For the phosphate-solubilizing ability, bacterial isolates were inoculated into 100 mL of optimum medium containing tricalcium phosphate at 35 • C. Quantitative spectrophotometric analysis of soluble phosphate was performed according to the described method of Kang et al. [57]. Alternatively, the pH was measured daily; the pH of the medium was recorded using pH meter equipped with a glass electrode. Furthermore, for bacterial identification, the 16S rRNA gene was amplified using general bacterial primers 27F and 1492R. The nucleotide sequences of PCR products were compared using the BLAST NCBI and EzTaxon program. A phylogenetic tree was constructed following the neighbor-joining method using MEGA v. 7 and was sent to the GenBank database for accession number. Isolate Mj1212 had phosphate-solubilizing activity and promotes mustard plant growth under normal conditions [58].

Plant Growth-Promoting Effect of Isolate AFFR02 and MJ1212 on Alfalfa under Normal and Post-drought Stress
Alfalfa seeds were purchased from Nature & Kids Korea Ltd. and sown in trays filled with autoclaved horticultural soil (which contained 51.5% coco peat, 10% peat moss, 13% vermiculite, 15% perlite, 10% zeolite, humic acid, 0.1% fertilizer, and 0.4% fungus-free bio-soil; Shinsung Mineral Co., Ltd., Goesan, Korea). After 2 weeks of germination, one seedling was transferred to each pot (100 mm diameter × 90 mm depth) and grown in a greenhouse. The experiment was conducted in the Kyungpook National University greenhouse, at Daegu, under natural light with controlled environmental conditions: day/night temperature: 28 ± 3 • C/25 ± 3 • C, with relative humidity of 60%-70%. The experiment was conducted in a completely randomized design, with each treatment replicated 10 times throughout the study. The experimental design included (a) no stress: (200 mL water per week), (b) 200 mL of cell suspension of isolate MJ1212 (2.75 × 10 7 CFU/mL) and AFFR02 (2.08 × 10 7 CFU/mL)/week), (c) drought stress: (50 mL water per week), and (d) bacterial-treated (50 mL cell suspension of isolate MJ1212 and AFFR02/week). After 2 weeks of drought stress, alfalfa plants were treated with normal water for 3 weeks (postdrought stress). The shoot/root length (SL/RL), shoot/root fresh weight (SFW/RFW), stalk diameter (SD), and CC were measured using a portable chlorophyll meter (SPAD-502, Konica, Japan). For chlorophyll, a and b and total carotenoids, the detailed method of Khan et al. [59] was followed using 80% acetone. Their content was measured spectrophotometrically at wavelengths of 663 nm, 465 nm, and 480 nm, respectively.

Determination of Leaf Water Potential and Electrolyte Leakage in Alfalfa Plants under Normal and Post-Drought-Stressed Conditions
Leaf relative water content (RWC) was measured according to a previously described method of Lubna et al. [60]. Individual leaves were collected and weighed (fresh weight; FW) and immersed in distilled water overnight. At the end, turgid weight (TW) was measured and kept in a preheated oven (75 • C for 48 h) to obtain dry weight (DW). The RWC was calculated using the formula: RWC % = ([FW − DW]/[TW − DW]) × 100. For electrolytic leakage (EL), 500 mg leaves were cut (5 mm), kept in 10 mL deionized water in a tube, and placed in a water bath at 32 • C. After 2 h, initial EC was measured using an EC1 m. Further plant samples were autoclaved (121 • C for 15 min), cooled (25 • C), and final EC2 was measured. EL was estimated using the following formula: EL = EC1 − EC2 × 100.

Endogenous Abscisic Acid Quantification in Alfalfa Plants under Normal and Post-Drought-Stressed Conditions
Endogenous ABA was quantified and extracted according to a method by Khan et al. [61] and Asaf et al. [62]. ABA was extracted from the aerial parts (freeze-dried plant samples, 0.3 g) with 30 mL extraction solution (95% isopropanol and 5% glacial acetic acid), and a chromatograph was run using 10 ng of Me-[2H6]-ABA standard. The suspension was filtered, and the filtrate was concentrated using a rotary evaporator. The residue was suspended in 4 mL of 1 N NaOH solution and rinsed three times with 3 mL of methylene chloride to eliminate traces of lipophilic materials. After decreasing the pH of the aqueous phase to 3.5 by adding 6N HCL, it was extracted by solvent extraction with ethyl acetate three times. The ethyl acetate extract was then evaporated, and dry residue was resuspended in a phosphate buffer solution (pH 8) and passed through the polyvinylpolypyrrolidone (PVPP) column. The eluted phosphate buffer solution was partitioned thrice with ethyl acetate (EtOAc) after adjusting the pH to 3.5 with 6N HCL. All three aliquots extracted were pooled and evaporated using a rotary evaporator. Furthermore, the fraction was methylated with diazomethane for detection, and ABA was quantified using gas chromatography-mass spectrometry (GC-MS) (6890N network gas chromatograph, Agilent Technologies). Software from ThermoQuest Corp., Manchester, UK, was used to monitor signal ions (m/z 162 and 190 for Me-ABA, and m/z 166 and 194 for Me-[2H6]-ABA).

Antioxidant Enzyme and Nonenzymatic Activities in Alfalfa Plants under Normal and Post-Drought-Stressed Conditions
The detailed method of Adhikari et al. [55] was followed to determine polyphenol content. Briefly, samples were extracted with 100% methanol and measured using a spectrophotometer (Shimadzu, Kyoto, Japan) at 750 nm. For flavonoid content, sample extracts were mixed with double distilled water and then NaNO 2 was added. After 5 min, 10% of 60 µL AlCl 3 and 1M NaOH was added and vortexed. The absorbance reading was taken at 500 nm using a spectrophotometer, as reported by Adhikari et al. [63]. Superoxide dismutase (SOD) was measured according to the detailed method of Lubna et al. [60]. SOD activity was expressed as enzyme unit (EU) nmol/g. For the DPPH-scavenging activity, a detailed method of Blois [64] was used with some modification. Absorption was measured at 517 nm using a spectrophotometer and calculated using the following equation: scavenging effect (%) = 1 − (Abs sample − Abs control ) × 100.

Determination of Mineral Uptake in Alfalfa Plants under Normal and Post-Drought -Stressed Conditions
Potassium (K), phosphorous (P), calcium (Ca), and magnesium (Mg) content in the shoots of bacterial-inoculated and non-inoculated post-drought alfalfa plants were investigated according to the detailed method of Sahile et al. [8] and Kang et al. [65], using an inductively coupled plasma mass spectrometer (ICP-MS; Optima 7900DV, Perkin-Elmer, Waltham, MA, USA).

Statistical Analysis
The results of this study were subjected to statistical analysis. The difference in the mean values was compared using Duncan's multiple range test using statistical analysis system (SAS) v.9.3. For graphical presentation, Graph Pad Prism was used. All experiments were conducted in triplicate.

Isolation and Screening
Alfalfa plants were collected from Seosan, Chungcheongnam-do Province, Korea. Sixteen rhizospheric bacterial strains were isolated from the roots of alfalfa plants and screened for phosphate-solubilizing activity and PEG stress tolerance. We used isolate Mj1212 with phosphate-solubilizing activity to promote mustard plant growth under normal conditions [58].

Screening for Polyethylene Glycol (PEG) Tolerance
All isolates, including Mj1212, were examined for their ability to grow in different PEG (0%, 5%, 10%, and 15%) concentration stress on LB media. The results from this investigation showed that under approximately 5% PEG, the growth of all isolates was normal, whereas at 10% PEG stress, bacterial growth declined. Only three isolates (AFFR02, AFFR07, and Mj1212) showed growth in 15% PEG LB media ( Figure S1).

Phosphate-Solubilizing Ability of Isolate AFFR02 and Mj1212
All isolates were screened for phosphate solubilization. The results showed that on National Botanical Research Institute's phosphate (NBRIP) media plates, the formation of clear halos indicates tricalcium phosphate solubilization capacity. Nine isolates were positive for phosphate solubilization ( Figure 1A). Therefore, the phosphate solubilization potential of selected isolates was cross-checked by monitoring the pH of bacterial-inoculated NBRIP media every 24 h ( Figure 1B). The pH results showed a decrease in response to phosphate-solubilizing activity of isolate AFFR02 and Mj1212 from an initial pH of 7.0 to 4.2 after 96 h. In contrast, the P-solubilizing curve showed an increase that confirms the phosphate-solubilizing activity of AFFR02 and Mj1212 in inoculated NBRIP liquid media ( Figure 1C).

Identification of Effective Rhizospheric Isolate
Based on their tolerance to PEG and having the highest potential of P-solubilizing characteristic, isolate AFFR02 was selected and identified. To find sequences similar to 16S rRNA gene sequence of isolate AFFR02, we checked the database of GenBank, NCBI, and EzTaxon. Identification results of 16S rRNA gene sequence of isolate AFFR01 showed close similarity to Enterobacter ludwigii (accession no: Kt261055). Furthermore, the selected sequence nucleotides were sent to the GenBank database and registered with accession number MW345827 for isolate AFFR02 ( Figure 2).

Identification of Effective Rhizospheric Isolate
Based on their tolerance to PEG and having the highest potential of P-solubilizing characteristic, isolate AFFR02 was selected and identified. To find sequences similar to 16S rRNA gene sequence of isolate AFFR02, we checked the database of GenBank, NCBI, and EzTaxon. Identification results of 16S rRNA gene sequence of isolate AFFR01 showed close similarity to Enterobacter ludwigii (accession no: Kt261055). Furthermore, the selected sequence nucleotides were sent to the GenBank database and registered with accession number MW345827 for isolate AFFR02 (Figure 2).

Effect of Relative Water Content, Electrolytic Leakage, and Endogenous Abscisic Acid Content on Alfalfa Plants under Normal and Post-Drought-Stressed Conditions
Water potential content of drought alfalfa showed a great influence in AFFR02-and Mj1212-inoculated plants. Under normal conditions, no significant differences were observed in bacterial-inoculated and control plants. However, in post-drought-stressed alfalfa plants, a significant decrease in RWC was observed (27.45%). In contrast, an increase in RWC in alfalfa post-drought-stressed plants was observed (24.06%) compared with post-drought-stressed alfalfa plants ( Figure 4A). Similarly, EC results showed a higher level in post-drought-stressed plants (68.87%) than in control plants. However, a decrease in EC in AFFR02-and Mj1212-inoculated alfalfa post-drought-stressed plants (19.82%) was observed compared with post-drought-stressed alfalfa plants ( Figure 4B). Furthermore, endogenous ABA regulation was observed in post-drought alfalfa plants in AFFR02and Mj1212-inoculated and non-inoculated plants. ABA results showed no difference under normal conditions; however, a significant increase in ABA content (164.42%) was observed in post-drought alfalfa plants. A significant decrease in ABA content (24.41%) in bacterial-inoculated plants was observed compared with control post-drought-stressed alfalfa plants ( Figure 4C).

Regulation of Antioxidants in Alfalfa Plants Inoculated with Isolate AFFR02 and Mj1212 under Normal and Post-Drought-Stressed Conditions
Different antioxidants, such as total phenolic content (TPC), DPPH, superoxide dismutase (SOD), and total flavonoid content (TFC) were observed in post-droughtstressed alfalfa, inoculated, and non-inoculated AFFR02 and Mj1212 plants ( Figure 5). TPC content was significantly higher in alfalfa post-drought-stressed AFFR02-and

Ion Uptake in Alfalfa Plants Inoculated with Isolate AFFR02 and Mj1212 under Normal and Post-Drought-Stressed Conditions
In this study, ICP analysis of potassium (K), phosphorous (P), calcium (Ca), and magnesium (Mg) was investigated in post-drought alfalfa AFFR02-and Mj1212inoculated plants (Figure 6). ICP results showed that a decrease in ion uptake; K (31.30%), P (24.85%), Ca (8.75%), and Mg (21.18%) content was observed in post-drought-stressed

Discussion
As previously reported in many studies, drought stress has been recognized as a critical environmental factor that limits plant development and metabolism [10,39,66]. The results of this study showed that plant biomass and growth attributes were significantly reduced under drought stress. These results are similar to the findings of Kusaka et al. [67], Chimenti et al. [68], and Erice et al. [69], indicating that drought stress causes a significant reduction in the quality of dry biomass of alfalfa, pearl millet, corn, and broccoli. This reduction in plant biomass may allow these cultivars to maintain RWC by decreasing the size of transpiring organs during drought stress [39]. The mutualistic interaction between plant-microbe interactions can enhance drought stress tolerance by enhancing growth performance. In this experiment, isolate AFFR02 and MJ1212 moderated the adverse effect of drought stress and improved plant growth and biomass content in post-drought alfalfa plants (Table 1; Figure 3). Enhancement in the growth attribute's (root/shoot length) biomass (fresh/DW) has been reported in several plants, such as wheat (Azospirillum sp. and Azospirillum brasilense), common bean (Paenibacillus

Discussion
As previously reported in many studies, drought stress has been recognized as a critical environmental factor that limits plant development and metabolism [10,39,66]. The results of this study showed that plant biomass and growth attributes were significantly reduced under drought stress. These results are similar to the findings of Kusaka et al. [67], Chimenti et al. [68], and Erice et al. [69], indicating that drought stress causes a significant reduction in the quality of dry biomass of alfalfa, pearl millet, corn, and broccoli. This reduction in plant biomass may allow these cultivars to maintain RWC by decreasing the size of transpiring organs during drought stress [39]. The mutualistic interaction between plant-microbe interactions can enhance drought stress tolerance by enhancing growth performance. In this experiment, isolate AFFR02 and MJ1212 moderated the adverse effect of drought stress and improved plant growth and biomass content in post-drought alfalfa plants (Table 1; Figure 3). Enhancement in the growth attribute's (root/shoot length) biomass (fresh/DW) has been reported in several plants, such as wheat (Azospirillum sp. and Azospirillum brasilense), common bean (Paenibacillus polymyxa and Rhizobium tropici), Arabidopsis (Pirifomospora indica), and broccoli (Variovorax sp.), inoculated with microbes under drought stress [11][12][13]32,35]. Similarly, CCs are also sensitive to drought stress, and a reduction in CC under drought stress has been reported in several plants, such as broccoli, cucumber, and tomato. In these experiments, CC decreased under drought stress (Table 2). However, enhancement in CC, Chl a, Chl b, and total carotenoid content were observed in post-drought alfalfa plants inoculated with isolate AFFR02 and MJ1212 ( Table 2). Enhancement in the photosynthetic rate and induced change in different ROSscavenging enzymes have been reported in plants inoculated with drought-tolerant isolates, such as Variovorax sp. (broccoli), Bacillus cereus, Bacillus subtilis, Serratia sp. (cucumber), Achromobacter piechaudii (tomato), and Bacillus sp. (corn), which helps the plants cope with drought stress [12,[70][71][72].
Drought stress affects nutrient uptake by impairing the translocation of some nutrients. This investigation of ICP analysis showed that drought stress negatively affects K, P, Ca, and Mg content in alfalfa ( Figure 6). These results agree with others, indicating that drought stress decreases Mg, P, and other nutrient content in alfalfa [39]. Enhancing nutrient availability through solubilization and chelation of minerals increased nutrient uptake efficiency [33]. P-solubilizing bacteria have attracted attention because of their agroeconomic biotechnological approach and can solubilize more phosphorus under stress [18,38]. Phosphorous (P) is a significant macronutrient required for various metabolic processes, such as photosynthesis and respiration. However, less P is available, and P-solubilizing bacteria can beneficially affect P uptake [18,38]. Results of P uptake showed that an increase in AFFR02-and Mj1212-inoculated alfalfa plants might be due to its P-solubilizing activity ( Figure 6B).
Similarly, potassium (K) plays an important role in the biochemical and physiological processes of plant growth, and survival of plants under stress [73]. Under drought stress, the diffusion rate of K in soil toward the roots decreases, depressing plant resistance to drought stress [73]. It was reported by Erdogan et al. [74] that strawberry plants inoculated with three PGPR (Paenibacillus polymyxa, Rhodococcus erythropois, and Pesudomonas fluorescens) mitigate drought stress and enhance nutrient uptake, including K. Furthermore, an increase in Ca and Mg content was observed in AFFR02-and Mj1212-inoculated alfalfa plants ( Figure 6C,D). It was reported that Ca and Mg playing a vital role in membrane protection, modulation of ions in the chloroplast, and postponing oxidative damage caused by drought stress [75,76]. In plants, 6%-25% of the total chlorophyll plays a vital role in photosynthesis and activation of many enzymes [77]. The decrease in chlorophyll content in post-drought-stressed alfalfa plants might be due to a decrease in Mg content ( Figure 6D).
Various environmental stresses, including drought stress, lead to decreased water availability and osmotic stress that promote the synthesis of endogenous ABA phytohormonal regulation and different antioxidant systems [23,78,79]. It was also observed that alfalfa plants inoculated with AFFR02 and MJ1212 induced drought stress tolerance by reducing water loss ( Figure 4A). This investigation showed that bacterial-inoculated plants had a higher RWC and lower EL than control post-drought-stressed alfalfa plants (Figure 4). A decrease in RWC and an increase in EL indicate a loss of turgor under drought stress, which results in less availability of water to plants [80,81]. In this investigation, lower EL and higher RWC was observed in bacterial-inoculated alfalfa postdrought-stressed plants. Our finding is also supported by other studies that reported that microbial-inoculated plants decrease stress, help fetch higher water content for plants, and decrease stomatal aperture [81][82][83][84]. Similarly, the decrease in the EL of post-drought alfalfa-inoculated plants mainly attribute to the integrity and stability of cellular tissue compared with drought-stressed plants. This increase in RWC and decrease in EL was also correlated with stomatal openings regulated by a complex hormonal network, such as ABA [35,84]. Since the discovery of ABA, several efforts have been devoted to understanding the synthesis of ABA under stress conditions [85][86][87]. Endogenous ABA level is significantly increased during drought stress by stimulating stomatal closure and adaptive physiological responses [12].
To further elucidate the role of microbes under post-drought stress in alfalfa plants, we investigate different antioxidant effects. Under stress conditions, an increase in damaging levels of ROS is a common factor [88,89]; however, to cope with ROS damage, plants induce enzymatic and nonenzymatic components [90]. SOD acts as a main enzymatic scavenger that scavenges superoxide (O 2 − ) and hydrogen peroxide (H 2 O 2 ) to H 2 O and O 2 [3,4,91]. This study showed that bacterial-inoculated alfalfa plants have higher SOD activities, suggesting inoculated microbe-induced drought tolerance in alfalfa plants ( Figure 5C). Furthermore, higher phenolic content, DPPH radicals, and total flavonoids were observed in bacterial-inoculated alfalfa post-drought-stressed plants compared with post-drought-stressed (untreated) plants ( Figure 5). This higher accumulation of total phenolic content is essential for maintaining the osmotic potential of plants, increasing physiological activities during drought stress [92]. Our results agree with the report of [92][93][94], who noted that PGPR induction increases phenolic content in wheat and pulse crops. Similarly, Singh et al. [95] and Asghari et al. [96] reported higher antioxidants, such as DPPH in inoculated rice and pennyroyal plants. Alternatively, other antioxidants' total flavonoids play a vital role by eliminating singlet oxygen and alleviating stress [93]. In bacterial-inoculated post-drought-stressed alfalfa plants, higher TFC content was observed ( Figure 5D). These results agree with the report of [93,97], who reported an increase in TFC content in Cariniana estrellensis and Cymbopogon citratus bacterial-inoculated drought-stressed plants.

Conclusions
This investigation showed that the local strain is closely related and identified as Enterobacter ludwigii. It was also observed that drought stress reduced the growth attributes of alfalfa. However, alfalfa plants inoculated with isolate AFFR02 and Mj1212 improved plant capacity to mitigate drought stress. Therefore, based on these results, further studies using genomic approaches to identify field trails of drought-stressed affected areas for their practical applications are needed.