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Alleviation of salt stress response in soybean plants with the endophytic bacterial isolate Curtobacterium sp. SAK1

Alleviation of salt stress response in soybean plants with the endophytic bacterial isolate... Background Salinity has been a major abiotic stressor that reduce the productivity. Previous studies reported that endophytic bacteria produce plant stress response hormones, antioxidants, and enzymes such as ACC deaminase. Augmentation of these metabolites and enzymes by endophytes mitigates the stress effects of salinity and improves plant growth and productivity. Methods Bacterial endophytes were isolated from Artemisia princeps Pamp, and evaluated for indole-3-acetic acid (IAA), abscisic acid (ABA), siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production and the ability to solubilize phosphate in the presence of NaCl (100–400 mM). SAK1 was applied to Glycine max cv. Pungsannamul to investigate salinity stress. Results Our results revealed that with an increase in NaCl concentration, the amount of ABA production in SAK1 increased, whereas IAA levels decreased. Bacterial ABA and JA degrade the reactive oxygen species and protect plants against stressors. Gas chromatography-mass spectrometry (GC-MS) analysis detected different gibberellins (GAs) and organic acids in SAK1. Interestingly, SAK1 inoculation significantly increased plant growth attributes under normal and salinity stress conditions, whereas a decrease in endogenous jasmonic acid and ABA content in the plants was recorded under salinity stress. IAA and GAs enhance number of root tips and hence improve nutrients uptake in plants. Polyphenolic oxidase and peroxidase were alleviated by elevated SAK1 in G. max plants under stress. ACC deaminase of SAK1 resulted deamination of ACC, up to −1 −1 330 nmol α-ketobutyrate mg h which could be a major reason of ethylene reduction promoting plant growth. Conclusion SAK1 relieved salinity stress in plants by producing different phytohormones, antioxidants, and ACC deaminase enzyme. SAK1 could be a new addition in batch of plant stress hormone-regulating endophytic bacteria that mitigates the effects of salt stress and promotes plant growth in G. max. . . . . Keywords Endophytic bacteria ACC deaminase Salt stress Phytohormones Antioxidant Introduction agricultural production, because they influence the yield of agricultural crops (Sgroy et al. 2009; Shrivastava and Kumar Various environmental stressors, including drought, high tem- 2015). Among these, soil salinization is one of the most severe perature, salinity, flood, insecticides, and soil pH, limit problems that has a negative impact on productivity and qual- ity and leads to reduction of the cultivated area. Salinity affects almost all aspects of plant growth; it imposes ionic toxicity, * In-Jung Lee osmotic stress, nutrient deficiency, and oxidative stress in ijlee@knu.ac.kr plants, and therefore, limits the absorption of soil water (Shrivastava and Kumar 2015). Around 20% of arable land School of Applied Biosciences, Kyungpook National University, and 33% of irrigated land worldwide are severely affected by Daegu 41566, Republic of Korea salinity stress (Ali et al. 2014; Pitman and Lauchli 2002; Natural and Medical Science Research Center, University of Nizwa, Sgroy et al. 2009). Owing to poor agricultural practices, ero- 616 Nizwa, Oman sion of rocks, and irrigation with salt water, there has been an Department of Biological Sciences, Faculty of Science, King annual increase of 10% in salinized areas (Shrivastava and Abdulaziz University, Jeddah, Saudi Arabia Kumar 2015). Salinity affects irrigated land, which produces Research Institute for Dok-do and Ulleung-do Island, Kyungpook 40% of the world’s food (Pimentel et al. 2004). Moreover, National University, Daegu, Republic of Korea 798 Ann Microbiol (2019) 69:797–808 Jamil et al. (2011) reported that more than 50% of the arable In the present study, the endophytic bacterial strain SAK1, land would be salinized by 2050. isolated from Artemisia princeps Pamp, was analyzed for the Extensive efforts and strategies have been used to reduce production of ACC deaminase, as well as phytohormones that the toxic effects of salinity stress on crop production; these promoted growth parameters of soybean plants under salinity include plant genetic engineering, the use of salt-resistant va- stress. rieties, salt stress-mitigating chemicals, and organic matter conditioners (Zhang et al. 2000). However, salinity is a chal- lenging problem for scientists to develop less expensive and Materials and methods viable approaches that are easy to adopt. The use of salt- tolerant microbes is an alternative method to improve crop Isolation of bacterial endophytes productivity. Recently, it has been shown that plant growth- promoting endophytic (PGPE) bacteria can help their host The endophytic bacteria were isolated using protocols de- plants cope with various biotic and abiotic stresses (El- scribed by Khan et al. (2014) from different plants growing Awady et al. 2015; Mayak et al. 2004; Sziderics et al. 2007; at Pohang beach (latitude 367′56.2′′N, longitude 12923′55.1′′ Yaish et al. 2015; Zhao et al. 2016). Besides the ability to E, and an elevation of 9.9 m), South Korea. The isolated produce phytohormones and secondary metabolites, several bacterial endophytes were screened on LB agar media. The 1-aminocyclopropane-1-carboxylate (ACC) deaminase- screened endophytic bacteria were incubated for 24 h at 28 °C, producing bacteria have been reported to promote the growth and pure colonies were observed for different morphological of plants under salt stress conditions (Cheng et al. 2007;Jalili features that included size, color, shape, and growth pattern, in et al. 2009). Such plant growth-promoting bacteria (PGPB) order to identify the bacteria. like Alcaligenes, Variovorax, Rhodococcus, Ochrobactrum, and Bacillus have been reported to produce ACC deaminase Indole acetic acid, siderophore production (Ahmad et al. 2011; Saravanakumar and Samiyappan 2007). and phosphate solubilization Under salinity stress, the plant hormone ethylene is accu- mulated in plants; however, bacteria containing ACC deami- The pure cultures of all bacterial isolates were screened for nase mitigate the production of stress ethylene by degrading indole acetic acid (IAA) and siderophore production, as well the higher amount of ACC produced in response to the stress- as their potential to solubilize phosphate. The method of ful conditions. This is because ACC deaminase cleaves ACC Patten and Glick (2002) was used for initial confirmation of to form ammonium and α-ketobutyrate, thereby decreasing IAA production by using the Salkowski reagent. The chemical ACC levels in plant tissues (Barnawal et al. 2016; Maxton method of Schwyn and Neilands (1987) was used for the et al. 2018). Previous reports have shown that ACC determination of siderophore production. Similarly, phosphate deaminase-producing PGPB can enhance growth in rice solubilization was evaluated following the method of (Jaemsaeng et al. 2018), tomato (Ali et al. 2014; Palaniyandi Katznelson and Bose (1959). et al. 2014), canola (Cheng et al. 2007; Jalili et al. 2009; Sergeeva et al. 2006), groundnut (Saravanakumar and Confirmation and quantification of ACC deaminase Samiyappan 2007), mung bean (Ahmad et al. 2011), musli activity in SAK1 (Barnawal et al. 2016), and piper (Maxton et al. 2018), under salt stress conditions. SAK1 was evaluated for the production of ACC deaminase by Soybean (Glycine max) is considered an important agricul- culturing it in Dworkin and Foster (DF) minimal medium for tural crop that is widely used as animal and human food, 20 h at 28 °C, in a shaking incubator at 200 rpm. The method because of its high oil (38%) and protein (18%) content of Penrose and Glick (2003) was used for the confirmation of (Guan et al. 2014). It is also known for its partial salt- ACC deaminase enzyme production by SAK1 in DF medium, sensitivity (Guan et al. 2014), causing a 20–40% reduction with slight modifications such as the incubation temperature in yield with increasing salinity stress (Papiernik et al. (28 °C) and centrifugation time (15 min). 2005). High salt stress has negative impacts on growth, seed For the quantification of ACC deaminase activity, the quality and quantity, and nodulation (Khan et al. 2018). In methods of Shaharoona et al. (2006) and Belimov et al. particular, the symbiotic relationship of rhizobia with legumes (2015) were used for the measurement of α-ketobutyrate is disturbed, causing cell dehydration and ion accumulation. produced upon the hydrolysis of ACC. The amount of α- Recent studies on the positive effects of microbes on plants ketobutyrate (μmol) produced by this reaction was deter- strongly recommend the use of microorganisms for the pro- mined by comparing the absorbance at 540 nm of the motion of plant growth and salt stress mitigation (El-Awady sample, to a standard curve of α-ketobutyrate. Here in et al. 2015; Sgroy et al. 2009; Szymanska et al. 2016; Yaish this reaction, the color of phenyl-hydrazine was devel- et al. 2015;Zhao etal. 2016). oped by the addition of 2 mL of a 2 M NaOH solution, Ann Microbiol (2019) 69:797–808 799 and after mixing, the absorbance of the mixture was mea- GC-MS-SIM analysis of isolates for in vitro IAA sured at 540 nm by using a spectrophotometer (Eppendorf and ABA production BioSpectrometer). To determine the ACC deaminase ac- tivity of SAK1, the isolate was grown in DF minimal SAK1 was grown in LB media with different NaCl concen- medium for 20 h at 28 °C; after harvesting the cells by trations (100 to 400 mM) for four consecutive days. LB me- centrifugation at 8000×g for 10 min, ACC deaminase ac- dia, containing 10 g tryptone, 5 g yeast extract, pH 7.0 ± 0.2, tivity was measured according to the method of Penrose was prepared and autoclaved. As the purpose was to examine and Glick (2003). In this method, the cell suspension IAA and ABA production dynamics, upon successful growth, without ACC was used as a negative control, and the culture was centrifuged (5000×g for 15 min). Culture (NH ) SO was used as a positive control. broth was analyzed for IAA and ABA, following previously 4 2 4 published methods (Khan et al. 2016;Qi etal. 1998). The concentration of IAA and ABA in the broths was calculated Bioassay assessment and molecular identification compared to known standards, by using gas chromatography- of isolates mass spectrometry (GC-MS) in selected ion monitoring mode (SIM). The bacterial isolates with diverse plant growth-promoting traits were selected for screening on rice. The sterilized seeds GC-MS-SIM analysis for the quantification of the rice cultivar ‘Waito-C’ (dwarf rice variety which is of gibberellins in bacterial isolates gibberellin-deficient mutant) were treated for 6 h with SAK1 9 −1 (10 cfu mL ) in a shaking incubator. Similar conditions were Selected strains of endophytic bacteria were cultured, centri- used for the untreated seeds as a control. Hoagland solution fuged (10,000×g), and filtered (45-μm filter paper) for quan- was used to grow the test seeds for 14 days under these con- tification of gibberellins (GAs). The isolated culture filtrate trolled environmental conditions: 14/10 h light/dark (light in- (CF) was analyzed for GAs through GC-MS-SIM. The quan- −2 −1 tensity of 250 μmol m s ), 28/24 °C, and 70% relative tification of GAs was performed according to the modified humidity. method of Khan et al. (2012). The standard protocol of Sambrook and Russell (2001) Different internal standards were used for GA analysis was used for the isolation of genomic DNA. Specific ([17, 17-2H ]. A C18 column (90–130 μm) was used for all primers 1492R (5′-CGG(T/C)TACCTTGTTACGACTT- extracts for different fractions. For each type of GA, the injec- 3′)and27F(5′-AGAGTTTGATC(C/A)TGGCTCAG-3′) tion volume of all aliquots was kept at 1 μL for GC-MS were used for the amplification of 16S rDNA, as sug- (Table 1). The amount of GAs (GA ,GA ,GA ,GA ,GA , 1 3 4 7 8 gested by (Khan et al. 2014). Furthermore, primers used GA ,GA ,GA ,GA ,GA ,and GA ) in the CF were 9 12 19 20 24 36 for the amplification of acdS genes were as follows: calculated from the peak area ratios. Similarly, retention times acdSf3, 5′-ATCGGCGGCATCCAGWSNAAYCANAC-3′ were determined by using the standards of hydrocarbon. and acdSr3,5 ′-GTGCATCGACTTGCCCTCRT ANACNGGRT-3′ (Lietal. 2015). The BLAST program of NCBI, GenBank database/EzTaxon, was used to deter- Analysis of organic acids in culture broth of bacterial mine the nucleotide sequence homology of the endophytic strains bacterial isolates. The Neighbor Joining (NJ) method was used for phylogenetic analysis through MEGA v. 6.1 The bacterial cultures were centrifuged (10,000×g) for 10 min (Tamura et al. 2013). followed by filtration of the supernatant through a 0.22-μm Table 1 Description of plants species and isolation of endophytes along with their number of yielded isolates having individual or multiple plant growth-promoting characteristics Plants name No of isolates Isolates having individual plant growth-promoting characteristics Isolates with multiple PGP characteristics IAA production Siderophore Phosphate Artemisia princeps Pamp. 24 16 5 6 8 Chenopodium ficifolium Smith. 6 1 0 2 0 Oenothera biennis L. 17 12 1 2 1 Echinochloa crus-galli (L.) Beauv. 12 7 1 3 4 800 Ann Microbiol (2019) 69:797–808 Millipore filter. A total of 10 μL filtratefrom eachsamplewas estimate the amount of endogenous JA. All treatments were injected into a high performance liquid chromatography sys- repeated in triplicate. tem (HPLC; Waters 600E), with the following conditions: column, RSpak KC-811 (8.0 × 300 mm); eluent, 0.1% Total proteins, peroxidase, polyphenol oxidase, −1 H PO /H O; flow rate, 1.0 mL min ; temperature, 40 °C. 3 4 2 and glutathione quantification The organic acid retention times and peak areas of chromato- grams were compared with standards from Sigma-Aldrich, For quantification of total proteins, the method of Bradford USA (Kang et al. 2012). All of the samples were analyzed (1976) was used, with slight modifications; the extracts were in triplicate. measured at 595 nm on a SHIMADZU spectrophotometer (Kyoto, Japan). Similarly, the antioxidant enzymes peroxidase Plant growth-promoting characteristics of bacterial (POD) and polyphenol oxidase (PPO) were also analyzed. isolates Briefly, the homogenized leaf samples (400 mg) were centri- fuged at 5000×g on 4 °C for 15 min. The method of Kar and The seeds of soybean cv. Pungsannamul were collected from Mishra (1976) was used for PPO and POD analyses, with Kyungpook National University’s Soybean Genetic Resource slight modifications. The final assay was determined at Centre, Republic of Korea. The seeds were tested for viability 420 nm for measuring POD and PPO. The method of and used in the present study. Surface sterilization of seeds Ellman (1959) was used for the determination of glutathione was performed with 2.5% sodium hypochlorite for 30 min, concentration. then rinsing with sterilized water. Upon the 10th day of seed germination in the trays, uniform plants were selected for fur- Statistical analysis ther processing. The autoclaved horticultural soil consisted of peat moss (10–15%), zeolite (6–8%), coco peat (45–50%), + −1 − All experiments were performed in triplicate and results col- perlite (35–40%), with NH ∼ 0.09 mg g ,NO ∼ 4 3 −1 −1 −1 lected were used for further analysis. A two-way ANOVAwas 0.205 mg g ,P O ∼ 0.35 mg g ,and K O ∼ 0.1 mg g . 2 5 2 used for statistical analysis, by using Bonferroni Post-Hoc test Plastic pots (10 cm × 10 cm × 9 cm) were used for the with a significance level ≤ 0.05. For comparing the effect of growth of soybean seedlings for 21 days consecutively, up PGPB on soybean growth and germination, a design that was to the V1 stage (vegetative stage of unifoliate node). Our ex- fully randomized was used. Graphpad Prism 5 (USA) was perimental design consisted of the following treatments: (a) used for graphical presentation and statistical analysis. control (normal soybean), (b) soybean with SAK1, (c) treat- ment 1 (100 mM NaCl) with or without SAK1, (d) treatment 2 (200 mM NaCl) with or without SAK1, and (e) treatment 3 (300 mM NaCl) with or without SAK1, in a growth chamber Results (24 h cycle: 28 °C for 14 h and 25 °C for 10 h with a relative humidity of 60 to 70%; Khan et al. 2018). For washing of A total of 59 endophytic bacterial strains were isolated from harvested cells, a 0.8% NaCl solution was used and the optical the roots of Oenothera biennis, Chenopodium ficifolium, density was adjusted to 0.5. Different growth parameters were A. princeps, and Echinochloa crus-galli plants, grown pre- analyzed, such as chlorophyll content, root and shoot length, dominantly in the Eastern sea coast on sand dunes at Pohang and fresh biomass of all seedlings. (Table 1). Biochemical and morphological tests were conduct- ed for determination of plant growth-promoting (PGP) traits. Quantification of endogenous phytohormones All isolates in this study showed one or more PGP traits, like in plants production of IAA, siderophores and solubilization of phos- phate. However, only 13 isolates showed two or three traits All plant samples were subjected to endogenous phytohor- (Table 1). Based on multiple PGP traits, five isolates were mone analysis and their quantification. The total endogenous subjected to further analysis on rice. The isolates that promot- ABA content was quantified according to the detailed method ed the largest increase in the weight of rice plants were select- of Qi et al. (1998), and each experiment was repeated in trip- ed for additional experiments. Our results on Waito-C rice licate. Similarly, the method of McCloud and Baldwin (1997) growth after inoculating selected isolates revealed that was used for quantification of endogenous jasmonic acid (JA) SAK1 remarkably increased rice growth (Fig. 1a) as com- content. All treatment samples (freeze-dried) were used for the pared to the other selected isolates and controls (Fig. 1a). extraction and quantification of JA (McCloud and Baldwin Similarly, acdS gene of the isolated bacterial endophyte was 1997). For these analyses, [9, 10-2H2]-9,10-dihydro-JA also amplified and confirmed by using specific primers, which (20 ng) was used as an internal standard. Furthermore, the revealed that SAK1 has the capability to produce ACC deam- peak areas were compared with their respective standards to inase (Fig. 1b). Ann Microbiol (2019) 69:797–808 801 Fig. 1 Growth parameters, siderophores, and 1-aminocyclopropane-1- carboxylate (ACC) deaminase activity. a Effect of different bacterial iso- lates on root and shoot lengths of rice. b Amplification of acdS gene of SAK1 Fig. 2 Characteristics of bacterial isolates. a Quantification of ACC (1- aminocyclopropane-1-carboxylate) deaminase production. b Indole acetic acid (IAA) content observed in culture broth (CB) of SAK1. Quantification of ACC deaminase and phytohormone SAK1 was grown in culture broth (CB) with high NaCl concentrations production of SAK1 (100, 200, 300, and 400 mM). c Content of abscisic acid (ABA) in CB of SAK1. SAK1 was grown in CB with high NaCl concentrations (100, 200, 300, and 400 mM). Each data point is the mean of three replicates. Error SAK1 was evaluated for the production of ACC deaminase bars represent standard errors. The bars represented with different letters and phytohormones. We estimated the capability of endophyt- are significantly different from each other as evaluated by DMRTanalysis ic SAK1 bacteria to produce ACC deaminase on DF minimal media containing its substrate ACC. SAK1 revealed the highest value of deamination of ACC, up to 330 nmol α- ABA is produced in response to higher saline stress condition. −1 −1 ketobutyrate mg h (Fig. 2a). This also suggests that the production of ABA may be advan- Moreover, SAK1 produce ABA and IAA in LB media in tageous for the growth of certain species under salinity and the presence of different NaCl concentrations (0, 100, 200, drought stress conditions. Conversely, the decreased amount 300, and 400 mM). Here, we noticed that the amount of of IAA content was measured under higher salt concentration ABA was rising steadily with the increase in concentration in the broth, compared to that in normal culture media (Fig. of salt in the media (Fig. 2c). Thus, the increased amount of 2b). Hence, the production of ABA is augmented in the 802 Ann Microbiol (2019) 69:797–808 presence of higher NaCl concentration while the amount of acid were produced at concentrations of 3.3, 2.5, and −1 IAA is lowered down in the LB medium. 0.66 μgmL ,respectively. SAK1 mitigates salinity stress Bioactive GAs detected in culture filtrate of SAK1 Soybean plants inoculated with SAK1 revealed interesting The culture filtrate of isolated SAK1 was analyzed for the results under salinity stress conditions. The plants treated with purpose of GAs. Results of SAK1 analysis showed different SAK1 greatly mitigated the adverse effects of salinity stress, components of GAs, e.g., GA ,GA ,GA ,andGA .The 7 8 24 36 and favored plant growth, compared to untreated stressed −1 quantities of GAs were as follows: GA , 0.847 ng mL ;GA , 7 8 plants (Table 2). The SAK1-treated plants under salt stress −1 −1 0.04 ng mL ;GA , 0.024 ng mL ;and GA , 24 36 had significantly enhanced length and biomass compared to −1 0.038 ng mL , as shown in Fig. 3a. those of untreated plants. Under normal conditions (Table 2), soybean plants inoculated with the SAK1 had greatly en- Detection of organic acids in SAK1 hanced root/shoot length and weight and chlorophyll content compared to those of control plants. Our results also revealed Results of organic acids analysis showed that the cultured that the growth of soybean was found to decrease when the salt concentration was increased, but SAK1 inoculation miti- filtrate of SAK1 was containing butyric acid, succinic acid, acetic acid, and quinic acid (Fig. 3b). SAK1 revealed the gated the negative effects of salt on soybean growth. This −1 showed that the beneficial effects of endophytic SAK1 inter- higher amount (8.2 μgmL ) of acetic acid compare to other organic acids. Malic acid was detected at a concentration of action mitigated salinity stress and promoted root/shoot −1 growth and chlorophyll content at different salt concentrations 3.9 μgmL , whereas quinic acid, succinic acid, and butyric compared to those in control plants. Endogenous ABA and JA content of soybean An increase in ABA levels was observed under different NaCl concentrations for the inoculated and non-inoculated soybean plants (Fig. 4a). However, the amount of endogenous ABA content under different salt concentrations is greatly reduced because of SAK1 inoculation. This demonstrates the stress- mitigating capability of endophytic SAK1. However, there was no significant difference in endogenous ABA levels be- tween control and unstressed SAK1-treated plants (Fig. 4a). Furthermore, our results revealed that higher salt stress en- hances endogenous JA content in soybean plants. However, SAK1 treatment lowered the amount of JA in treated plants, when the results were compared to those of controls (Fig. 4b). Effect of SAK1 on antioxidant activities and total protein content of soybean The protein level increases proportionally to the increase in salt concentration in soybean (Fig. 5a). However, plants treat- ed with SAK1 showed a reduced amount of total protein at different salt concentrations. The application of SAK1 en- hanced total protein content in stressed conditions, compared to control plants (Fig. 5a). It has already been shown that reactive oxygen species Fig. 3 a Gas chromatography-mass spectrometry (GC-MS-SIM) analysis (ROS) are produced during elevated NaCl concentrations that and quantification of different gibberellins (GAs) by comparing them leads to oxidative stress of plants (Habib et al. 2016). with the internal standard. b Organic acids (low molecular weight) pro- Consequently, we also inspected the antioxidant enzyme ac- duced by SAK1. HPLC analysis and detection of organic acids used, in tivities in the present study (Fig. 5). Our results revealed that relation to their respective standards. Given letter(s) are significantly dif- ferent at the 5% level by DMRT salt stress significantly increased the activity of PPO in Ann Microbiol (2019) 69:797–808 803 Table 2 Effect of bacterial isolates on growth attributes of the plant exposed to different concentrations of salts Treatments SL (cm) RL (cm) SFW (g) RFW (g) CC (SPAD) Control 19.52 ± 2.2ab 16.94 ± 0.8abc 2.11 ± 0.3b 2.17 ± 0.4b 31.83 ± 0.9ab Isolate SAK1 22.04 ± 2.3a 18.66 ± 0.5a 2.27 ± 0.1a 2.60 ± 0.3a 33.03 ± 1.5a 100 mM NaCl 17.61 ± 2.0bc 16.50 ± 0.5bc 1.92 ± 0.3c 1.94 ± 0.4cb 30.69 ± 1.1bc 100 mM NaCl + SAK1 19.15 ± 2.1ab 18.13 ± 1.4ab 2.09 ± 0.4b 2.15 ± 0.3b 32.03 ± 1.0ab 200 mM NaCl 15.03 ± 4.1 cd 14.83 ± 0.9 dc 1.77 ± 0.3d 1.73 ± 0.5 cd 29.06 ± 1.2c 200 mM NaCl + SAK1 17.84 ± 2.9bc 16.36 ± 1.0bc 1.95 ± 0.5c 1.95 ± 0.4cb 31.46 ± 1.7ab 300 mM NaCl 12.61 ± 2.3d 12.86 ± 1.5d 0.84 ± 0.2f 1.40 ± 0.4e 27.96 ± 1.4d 300 mM NaCl + SAK1 15.60 ± 0.5bcd 13.36 ± 1.6d 1.17 ± 0.4e 1.46 ± 0.5de 28.50 ± 1.5c uninoculated plants, compared to that in SAK1-treated plants of POD in soybean plants under salt stress, compared to that in (Fig. 5b). Furthermore, SAK1 greatly reduced PPO activity in uninoculated plants (Fig. 5c). Contrary to this, the initial ap- soybean plants treated with different salt concentrations (100 plication of SAK1 induced POD activity to a large extent to 300 mM). However, no notable difference was observed compared to that in control unstressed plants (Fig. 5c). between inoculated and non-inoculated control plants (Fig. Reduced glutathione is a central cellular antioxidant and 5b). signaling compound that stimulates many vital cellular pro- As shown in Fig. 6c, the enzymatic activity of POD was cesses. Exposure to different concentration of salt stress enhanced in the plants, similar to that of total protein content, caused an increased formation of ROS and leads to oxidative under high salinity stress. SAK1 greatly arrested the activity stress. Our results demonstrated that glutathione concentra- tions were slightly higher in SAK1-inoculated soybean plants under salinity stress (Fig. 5d). In addition, a decrease in glu- tathione concentration was observed in soybean at all NaCl concentrations tested (100, 200, and 300 mM). It appears that no notable differences were observed in glutathione concen- trations between SAK1-inoculated and control soybean plants (Fig. 5d). The increase in glutathione content implicates a response to oxidative stress, to enhance plant growth and ame- liorate salt stress. Identification and phylogenetic analysis of SAK1 Molecular identification and phylogenetic analysis of SAK1 were performed by amplifying and sequencing the 16S rRNA gene and comparing it to a database of known 16S rRNA sequences. The results revealed that SAK1 exhibited a high level of 16S rRNA sequence identity (99.93%) with Curtobacterium oceanosedimentum ATCC31317 (T:superscript) (Fig. 6). The sequence was submitted to NCBI with the accession number MF949056. Discussion SAK1 was employed in the present study as salinity stress Fig. 4 Jasmonic acid (JA) and abscisic acid (ABA) content quantification relevant to enhance the plant tolerance against high salt in soybean. a Effect of SAK1 on the ABA content in soybean growing in high NaCl concentrations (100, 200, and 300 mM). b Effect of SAK1 on concentration. Biochemical analysis of SAK1 showed the the content of JA in soybean growing in high NaCl concentrations (100, bacterial strain produced different phytohormones such as 200, and 300 mM). Each data point is the mean of three replicates. Error ABA, JA, IAA, GAs, and antioxidants including polyphe- bars represent standard errors. The columns represented with different nolic oxidase, peroxidase, and reduced glutathione. The letters are significantly different from each other as evaluated by DMRT analysis SAK1 also produced different organic acids and ACC 804 Ann Microbiol (2019) 69:797–808 Fig. 5 Assessment of peroxidase, polyphenol oxidase, and total proteins in soybean. Effect of SAK1 on a total protein content, b polyphenol oxidase (PPO), c peroxidase, and d reduced glutathione activities in plants exposed to high concentrations of NaCl (SS1 = salt stress 1, SS2 = salt stress 2, SS3 = salt stress 3). Data are expressed as the mean of three replicates. Error bars represent standard errors, with different letters indicating statistically significant differences deaminase enzyme. Phytohormones, e.g., IAA and GAs, root and shoot growth. Bacterial ACC deaminase de- have been known growth hormones which enhance shoot grades ACC and reduces the deleterious effect of ethyl- length, root length, and number of root tips, leading to ene, ameliorating plant stress and promoting plant growth improve nutrients uptake and hence improve plant health (Cheng et al. 2007). Inoculation bacteria like SAK1 pro- under stress conditions (Ullah et al. 2013). ABA and JA ducing ACC deaminase induce longer roots which might are common stress response phytohormones which de- be helpful in the uptake of relatively more water soil un- grade the reactive oxygen species and protect plants from der drought/salinity stress conditions, thus increasing wa- the hazardous effects of stressors. In addition, antioxi- ter use efficiency of the plants under salinity (Shaharoona dants produced by SAK1 are linked with oxidative stress et al. 2006). tolerance. Ethylene is a gaseous plant growth regulator PGPB that produce ACC deaminase and phytohor- which regulates plant homoeostasis and results in reduced mones belong to various bacterial genera, including Fig. 6 Phylogenetic tree of SAK1 was constructed 16S rRNA sequences using neighbor joining (NJ) and maximum likelihood methods. Numbers above the branches represent the bootstrap values. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree Ann Microbiol (2019) 69:797–808 805 Arthrobacter, Microbacterium, Bacillus, Burkholderia, cellular toxicity in plants (Hasegawa et al. 2000; Mittova Rhizobium, Klebsiella,and Pseudomonas, that are known et al. 2004). To counteract adverse effects of ROS and oxida- to improve crop growth and enhance plant tolerance to tive stress, plants activate their antioxidant defense machinery, various abiotic and biotic stresses (Ali et al. 2018;Ali such as superoxide dismutase (SOD), POD, catalase (CAT), and Kim 2018; Egamberdieva et al. 2015; Gamalero and glutathione reductase (GR), that protect the plant against et al. 2010; Grover et al. 2011;Yue et al. 2007) including cellular stress and scavenge excess ROS (Kim et al. 2005). In salinity. Plant ACC is a main component of ethylene bio- the present study, SAK1-inoculated soybean plants showed synthesis which is a stress hormone produced during lower ROS levels, and the antioxidant enzyme activities (TP, stress and damages the plant tissues. The strain SAK1 PPO, and POD) were considerably reduced compared to those ACC deaminase degrades ACC and reduces the deleteri- of control soybean plants growing under different salinity ous effect of ethylene, ameliorating plant stress and pro- levels. Similar results were reported for lettuce, potato, and moting plant growth (Sergeeva et al. 2006). okra, where PGPB decreased the activities of ROS under in- Previous studies reported that salinity stress affects bio- creasing salinity stress (Gururani et al. 2013; Habib et al. chemical, morphological, and physiological functions in crop 2016; Hyo Shim Han 2005). plants (Shrivastava and Kumar 2015). Glutathione is an important antioxidant that regulates stress In this study, NaCl suppressed plant growth, and with in- responses by reacting with ROS and maintaining an intracel- creasing salt concentrations, a higher decline in plant growth lular redox state; it also enhances growth and salt tolerance in was observed (Table 2). However, with the help of SAK1, the plants (Chen et al. 2012; Csiszár et al. 2014). When intracel- adverse influence of NaCl stress on plant growth could be lular GSH concentrations are reduced, plants repeatedly dem- alleviated, by increasing biomass and chlorophyll content, onstrate a decrease in oxidative stress sensitivity (Grant et al. compared to control plants (Table 2). This clearly demon- 1997; Kushnir et al. 1995). In the present study, concentra- strates that SAK1 alleviates the debilitating effects of salt tions of glutathione were found to be slightly higher in SAK1- stress. Irizarry and White (2017) reported that inoculated soybean plants than in non-inoculated plants. It has Curtobacterium oceanosedimentum produce IAA and been previously reported that glutathione regulates biotic and enhanced the growth of cotton plant under salinity stress. abiotic stress responses, and plays a pivotal role in the photo- Similarly, Bianco and Defez (2009, 2010) reported that in synthetic regulation of plants (Diaz-Vivancos et al. 2015; Gill saline environment IAA-producing bacteria also increase root et al. 2013). In SAK1-inoculated soybean plants, increases in and shoot length of chickpea and Medicago plants, compare to glutathione levels were observed compared to those in non- uninoculated plants. inoculated plants, which indicated a comparatively high po- Abscisic acid (ABA), a phytohormone important for tential of ROS scavenging that increased photosynthesis, the growth and development of plants, plays a vital role growth, and shoot biomass. Our findings support the results in many stress signaling pathways, and is a notable plant of Fatma et al. (2014), who also found that an increase in stress marker. Plants adjust ABA levels constantly glutathione levels during salt stress could improve growth through stomatal closure, minimizing water loss and ac- and photosynthesis in plants. tivating many stress-responsive genes, in response to Together, our results lead to the conclusion that the appli- changing physiological and environmental conditions cation of Curtobacterium sp. SAK1 greatly mitigates the ef- (Zhang et al. 2006). Salinity stress leads to an increase fects of salt stress and promotes plant growth in G. max. in ABA content (Wang et al. 2001). Our findings show Authors’ contributions MAK, SA, SMK, and SA conducted the exper- that plants inoculated with SAK1 significantly produce iments. ALK and IU helped in writing of the manuscript. IJL designed, lowers endogenous ABA production compared to non- supervised, and financed the research. All authors have read and agreed to inoculated plants (Fig. 4a). The production of ABA by its content and also that the manuscript conforms to the journal’spolicies. PGPB improves growth of the plant and mitigates salt stress (Cohen et al. 2008). Suppressive influence of sa- Funding This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) linity stress on plant germination has been associated funded by the Ministry of Education (2016R1A6A1A05011910). with a reduced level of endogenous hormones. Endophytic originated plant growth regulators have been Compliance with ethical standards found to have same effects in plant as of exogenous phytohormones (Ullah et al. 2019). In this regard, the Conflicts of interest The authors declare that they have no conflict of SAK1, used in present study, could be a new addition interest. in batch of plant stress hormone-regulating endophytic bacteria (PSHEB). 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Alleviation of salt stress response in soybean plants with the endophytic bacterial isolate Curtobacterium sp. SAK1

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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Abstract

Background Salinity has been a major abiotic stressor that reduce the productivity. Previous studies reported that endophytic bacteria produce plant stress response hormones, antioxidants, and enzymes such as ACC deaminase. Augmentation of these metabolites and enzymes by endophytes mitigates the stress effects of salinity and improves plant growth and productivity. Methods Bacterial endophytes were isolated from Artemisia princeps Pamp, and evaluated for indole-3-acetic acid (IAA), abscisic acid (ABA), siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production and the ability to solubilize phosphate in the presence of NaCl (100–400 mM). SAK1 was applied to Glycine max cv. Pungsannamul to investigate salinity stress. Results Our results revealed that with an increase in NaCl concentration, the amount of ABA production in SAK1 increased, whereas IAA levels decreased. Bacterial ABA and JA degrade the reactive oxygen species and protect plants against stressors. Gas chromatography-mass spectrometry (GC-MS) analysis detected different gibberellins (GAs) and organic acids in SAK1. Interestingly, SAK1 inoculation significantly increased plant growth attributes under normal and salinity stress conditions, whereas a decrease in endogenous jasmonic acid and ABA content in the plants was recorded under salinity stress. IAA and GAs enhance number of root tips and hence improve nutrients uptake in plants. Polyphenolic oxidase and peroxidase were alleviated by elevated SAK1 in G. max plants under stress. ACC deaminase of SAK1 resulted deamination of ACC, up to −1 −1 330 nmol α-ketobutyrate mg h which could be a major reason of ethylene reduction promoting plant growth. Conclusion SAK1 relieved salinity stress in plants by producing different phytohormones, antioxidants, and ACC deaminase enzyme. SAK1 could be a new addition in batch of plant stress hormone-regulating endophytic bacteria that mitigates the effects of salt stress and promotes plant growth in G. max. . . . . Keywords Endophytic bacteria ACC deaminase Salt stress Phytohormones Antioxidant Introduction agricultural production, because they influence the yield of agricultural crops (Sgroy et al. 2009; Shrivastava and Kumar Various environmental stressors, including drought, high tem- 2015). Among these, soil salinization is one of the most severe perature, salinity, flood, insecticides, and soil pH, limit problems that has a negative impact on productivity and qual- ity and leads to reduction of the cultivated area. Salinity affects almost all aspects of plant growth; it imposes ionic toxicity, * In-Jung Lee osmotic stress, nutrient deficiency, and oxidative stress in ijlee@knu.ac.kr plants, and therefore, limits the absorption of soil water (Shrivastava and Kumar 2015). Around 20% of arable land School of Applied Biosciences, Kyungpook National University, and 33% of irrigated land worldwide are severely affected by Daegu 41566, Republic of Korea salinity stress (Ali et al. 2014; Pitman and Lauchli 2002; Natural and Medical Science Research Center, University of Nizwa, Sgroy et al. 2009). Owing to poor agricultural practices, ero- 616 Nizwa, Oman sion of rocks, and irrigation with salt water, there has been an Department of Biological Sciences, Faculty of Science, King annual increase of 10% in salinized areas (Shrivastava and Abdulaziz University, Jeddah, Saudi Arabia Kumar 2015). Salinity affects irrigated land, which produces Research Institute for Dok-do and Ulleung-do Island, Kyungpook 40% of the world’s food (Pimentel et al. 2004). Moreover, National University, Daegu, Republic of Korea 798 Ann Microbiol (2019) 69:797–808 Jamil et al. (2011) reported that more than 50% of the arable In the present study, the endophytic bacterial strain SAK1, land would be salinized by 2050. isolated from Artemisia princeps Pamp, was analyzed for the Extensive efforts and strategies have been used to reduce production of ACC deaminase, as well as phytohormones that the toxic effects of salinity stress on crop production; these promoted growth parameters of soybean plants under salinity include plant genetic engineering, the use of salt-resistant va- stress. rieties, salt stress-mitigating chemicals, and organic matter conditioners (Zhang et al. 2000). However, salinity is a chal- lenging problem for scientists to develop less expensive and Materials and methods viable approaches that are easy to adopt. The use of salt- tolerant microbes is an alternative method to improve crop Isolation of bacterial endophytes productivity. Recently, it has been shown that plant growth- promoting endophytic (PGPE) bacteria can help their host The endophytic bacteria were isolated using protocols de- plants cope with various biotic and abiotic stresses (El- scribed by Khan et al. (2014) from different plants growing Awady et al. 2015; Mayak et al. 2004; Sziderics et al. 2007; at Pohang beach (latitude 367′56.2′′N, longitude 12923′55.1′′ Yaish et al. 2015; Zhao et al. 2016). Besides the ability to E, and an elevation of 9.9 m), South Korea. The isolated produce phytohormones and secondary metabolites, several bacterial endophytes were screened on LB agar media. The 1-aminocyclopropane-1-carboxylate (ACC) deaminase- screened endophytic bacteria were incubated for 24 h at 28 °C, producing bacteria have been reported to promote the growth and pure colonies were observed for different morphological of plants under salt stress conditions (Cheng et al. 2007;Jalili features that included size, color, shape, and growth pattern, in et al. 2009). Such plant growth-promoting bacteria (PGPB) order to identify the bacteria. like Alcaligenes, Variovorax, Rhodococcus, Ochrobactrum, and Bacillus have been reported to produce ACC deaminase Indole acetic acid, siderophore production (Ahmad et al. 2011; Saravanakumar and Samiyappan 2007). and phosphate solubilization Under salinity stress, the plant hormone ethylene is accu- mulated in plants; however, bacteria containing ACC deami- The pure cultures of all bacterial isolates were screened for nase mitigate the production of stress ethylene by degrading indole acetic acid (IAA) and siderophore production, as well the higher amount of ACC produced in response to the stress- as their potential to solubilize phosphate. The method of ful conditions. This is because ACC deaminase cleaves ACC Patten and Glick (2002) was used for initial confirmation of to form ammonium and α-ketobutyrate, thereby decreasing IAA production by using the Salkowski reagent. The chemical ACC levels in plant tissues (Barnawal et al. 2016; Maxton method of Schwyn and Neilands (1987) was used for the et al. 2018). Previous reports have shown that ACC determination of siderophore production. Similarly, phosphate deaminase-producing PGPB can enhance growth in rice solubilization was evaluated following the method of (Jaemsaeng et al. 2018), tomato (Ali et al. 2014; Palaniyandi Katznelson and Bose (1959). et al. 2014), canola (Cheng et al. 2007; Jalili et al. 2009; Sergeeva et al. 2006), groundnut (Saravanakumar and Confirmation and quantification of ACC deaminase Samiyappan 2007), mung bean (Ahmad et al. 2011), musli activity in SAK1 (Barnawal et al. 2016), and piper (Maxton et al. 2018), under salt stress conditions. SAK1 was evaluated for the production of ACC deaminase by Soybean (Glycine max) is considered an important agricul- culturing it in Dworkin and Foster (DF) minimal medium for tural crop that is widely used as animal and human food, 20 h at 28 °C, in a shaking incubator at 200 rpm. The method because of its high oil (38%) and protein (18%) content of Penrose and Glick (2003) was used for the confirmation of (Guan et al. 2014). It is also known for its partial salt- ACC deaminase enzyme production by SAK1 in DF medium, sensitivity (Guan et al. 2014), causing a 20–40% reduction with slight modifications such as the incubation temperature in yield with increasing salinity stress (Papiernik et al. (28 °C) and centrifugation time (15 min). 2005). High salt stress has negative impacts on growth, seed For the quantification of ACC deaminase activity, the quality and quantity, and nodulation (Khan et al. 2018). In methods of Shaharoona et al. (2006) and Belimov et al. particular, the symbiotic relationship of rhizobia with legumes (2015) were used for the measurement of α-ketobutyrate is disturbed, causing cell dehydration and ion accumulation. produced upon the hydrolysis of ACC. The amount of α- Recent studies on the positive effects of microbes on plants ketobutyrate (μmol) produced by this reaction was deter- strongly recommend the use of microorganisms for the pro- mined by comparing the absorbance at 540 nm of the motion of plant growth and salt stress mitigation (El-Awady sample, to a standard curve of α-ketobutyrate. Here in et al. 2015; Sgroy et al. 2009; Szymanska et al. 2016; Yaish this reaction, the color of phenyl-hydrazine was devel- et al. 2015;Zhao etal. 2016). oped by the addition of 2 mL of a 2 M NaOH solution, Ann Microbiol (2019) 69:797–808 799 and after mixing, the absorbance of the mixture was mea- GC-MS-SIM analysis of isolates for in vitro IAA sured at 540 nm by using a spectrophotometer (Eppendorf and ABA production BioSpectrometer). To determine the ACC deaminase ac- tivity of SAK1, the isolate was grown in DF minimal SAK1 was grown in LB media with different NaCl concen- medium for 20 h at 28 °C; after harvesting the cells by trations (100 to 400 mM) for four consecutive days. LB me- centrifugation at 8000×g for 10 min, ACC deaminase ac- dia, containing 10 g tryptone, 5 g yeast extract, pH 7.0 ± 0.2, tivity was measured according to the method of Penrose was prepared and autoclaved. As the purpose was to examine and Glick (2003). In this method, the cell suspension IAA and ABA production dynamics, upon successful growth, without ACC was used as a negative control, and the culture was centrifuged (5000×g for 15 min). Culture (NH ) SO was used as a positive control. broth was analyzed for IAA and ABA, following previously 4 2 4 published methods (Khan et al. 2016;Qi etal. 1998). The concentration of IAA and ABA in the broths was calculated Bioassay assessment and molecular identification compared to known standards, by using gas chromatography- of isolates mass spectrometry (GC-MS) in selected ion monitoring mode (SIM). The bacterial isolates with diverse plant growth-promoting traits were selected for screening on rice. The sterilized seeds GC-MS-SIM analysis for the quantification of the rice cultivar ‘Waito-C’ (dwarf rice variety which is of gibberellins in bacterial isolates gibberellin-deficient mutant) were treated for 6 h with SAK1 9 −1 (10 cfu mL ) in a shaking incubator. Similar conditions were Selected strains of endophytic bacteria were cultured, centri- used for the untreated seeds as a control. Hoagland solution fuged (10,000×g), and filtered (45-μm filter paper) for quan- was used to grow the test seeds for 14 days under these con- tification of gibberellins (GAs). The isolated culture filtrate trolled environmental conditions: 14/10 h light/dark (light in- (CF) was analyzed for GAs through GC-MS-SIM. The quan- −2 −1 tensity of 250 μmol m s ), 28/24 °C, and 70% relative tification of GAs was performed according to the modified humidity. method of Khan et al. (2012). The standard protocol of Sambrook and Russell (2001) Different internal standards were used for GA analysis was used for the isolation of genomic DNA. Specific ([17, 17-2H ]. A C18 column (90–130 μm) was used for all primers 1492R (5′-CGG(T/C)TACCTTGTTACGACTT- extracts for different fractions. For each type of GA, the injec- 3′)and27F(5′-AGAGTTTGATC(C/A)TGGCTCAG-3′) tion volume of all aliquots was kept at 1 μL for GC-MS were used for the amplification of 16S rDNA, as sug- (Table 1). The amount of GAs (GA ,GA ,GA ,GA ,GA , 1 3 4 7 8 gested by (Khan et al. 2014). Furthermore, primers used GA ,GA ,GA ,GA ,GA ,and GA ) in the CF were 9 12 19 20 24 36 for the amplification of acdS genes were as follows: calculated from the peak area ratios. Similarly, retention times acdSf3, 5′-ATCGGCGGCATCCAGWSNAAYCANAC-3′ were determined by using the standards of hydrocarbon. and acdSr3,5 ′-GTGCATCGACTTGCCCTCRT ANACNGGRT-3′ (Lietal. 2015). The BLAST program of NCBI, GenBank database/EzTaxon, was used to deter- Analysis of organic acids in culture broth of bacterial mine the nucleotide sequence homology of the endophytic strains bacterial isolates. The Neighbor Joining (NJ) method was used for phylogenetic analysis through MEGA v. 6.1 The bacterial cultures were centrifuged (10,000×g) for 10 min (Tamura et al. 2013). followed by filtration of the supernatant through a 0.22-μm Table 1 Description of plants species and isolation of endophytes along with their number of yielded isolates having individual or multiple plant growth-promoting characteristics Plants name No of isolates Isolates having individual plant growth-promoting characteristics Isolates with multiple PGP characteristics IAA production Siderophore Phosphate Artemisia princeps Pamp. 24 16 5 6 8 Chenopodium ficifolium Smith. 6 1 0 2 0 Oenothera biennis L. 17 12 1 2 1 Echinochloa crus-galli (L.) Beauv. 12 7 1 3 4 800 Ann Microbiol (2019) 69:797–808 Millipore filter. A total of 10 μL filtratefrom eachsamplewas estimate the amount of endogenous JA. All treatments were injected into a high performance liquid chromatography sys- repeated in triplicate. tem (HPLC; Waters 600E), with the following conditions: column, RSpak KC-811 (8.0 × 300 mm); eluent, 0.1% Total proteins, peroxidase, polyphenol oxidase, −1 H PO /H O; flow rate, 1.0 mL min ; temperature, 40 °C. 3 4 2 and glutathione quantification The organic acid retention times and peak areas of chromato- grams were compared with standards from Sigma-Aldrich, For quantification of total proteins, the method of Bradford USA (Kang et al. 2012). All of the samples were analyzed (1976) was used, with slight modifications; the extracts were in triplicate. measured at 595 nm on a SHIMADZU spectrophotometer (Kyoto, Japan). Similarly, the antioxidant enzymes peroxidase Plant growth-promoting characteristics of bacterial (POD) and polyphenol oxidase (PPO) were also analyzed. isolates Briefly, the homogenized leaf samples (400 mg) were centri- fuged at 5000×g on 4 °C for 15 min. The method of Kar and The seeds of soybean cv. Pungsannamul were collected from Mishra (1976) was used for PPO and POD analyses, with Kyungpook National University’s Soybean Genetic Resource slight modifications. The final assay was determined at Centre, Republic of Korea. The seeds were tested for viability 420 nm for measuring POD and PPO. The method of and used in the present study. Surface sterilization of seeds Ellman (1959) was used for the determination of glutathione was performed with 2.5% sodium hypochlorite for 30 min, concentration. then rinsing with sterilized water. Upon the 10th day of seed germination in the trays, uniform plants were selected for fur- Statistical analysis ther processing. The autoclaved horticultural soil consisted of peat moss (10–15%), zeolite (6–8%), coco peat (45–50%), + −1 − All experiments were performed in triplicate and results col- perlite (35–40%), with NH ∼ 0.09 mg g ,NO ∼ 4 3 −1 −1 −1 lected were used for further analysis. A two-way ANOVAwas 0.205 mg g ,P O ∼ 0.35 mg g ,and K O ∼ 0.1 mg g . 2 5 2 used for statistical analysis, by using Bonferroni Post-Hoc test Plastic pots (10 cm × 10 cm × 9 cm) were used for the with a significance level ≤ 0.05. For comparing the effect of growth of soybean seedlings for 21 days consecutively, up PGPB on soybean growth and germination, a design that was to the V1 stage (vegetative stage of unifoliate node). Our ex- fully randomized was used. Graphpad Prism 5 (USA) was perimental design consisted of the following treatments: (a) used for graphical presentation and statistical analysis. control (normal soybean), (b) soybean with SAK1, (c) treat- ment 1 (100 mM NaCl) with or without SAK1, (d) treatment 2 (200 mM NaCl) with or without SAK1, and (e) treatment 3 (300 mM NaCl) with or without SAK1, in a growth chamber Results (24 h cycle: 28 °C for 14 h and 25 °C for 10 h with a relative humidity of 60 to 70%; Khan et al. 2018). For washing of A total of 59 endophytic bacterial strains were isolated from harvested cells, a 0.8% NaCl solution was used and the optical the roots of Oenothera biennis, Chenopodium ficifolium, density was adjusted to 0.5. Different growth parameters were A. princeps, and Echinochloa crus-galli plants, grown pre- analyzed, such as chlorophyll content, root and shoot length, dominantly in the Eastern sea coast on sand dunes at Pohang and fresh biomass of all seedlings. (Table 1). Biochemical and morphological tests were conduct- ed for determination of plant growth-promoting (PGP) traits. Quantification of endogenous phytohormones All isolates in this study showed one or more PGP traits, like in plants production of IAA, siderophores and solubilization of phos- phate. However, only 13 isolates showed two or three traits All plant samples were subjected to endogenous phytohor- (Table 1). Based on multiple PGP traits, five isolates were mone analysis and their quantification. The total endogenous subjected to further analysis on rice. The isolates that promot- ABA content was quantified according to the detailed method ed the largest increase in the weight of rice plants were select- of Qi et al. (1998), and each experiment was repeated in trip- ed for additional experiments. Our results on Waito-C rice licate. Similarly, the method of McCloud and Baldwin (1997) growth after inoculating selected isolates revealed that was used for quantification of endogenous jasmonic acid (JA) SAK1 remarkably increased rice growth (Fig. 1a) as com- content. All treatment samples (freeze-dried) were used for the pared to the other selected isolates and controls (Fig. 1a). extraction and quantification of JA (McCloud and Baldwin Similarly, acdS gene of the isolated bacterial endophyte was 1997). For these analyses, [9, 10-2H2]-9,10-dihydro-JA also amplified and confirmed by using specific primers, which (20 ng) was used as an internal standard. Furthermore, the revealed that SAK1 has the capability to produce ACC deam- peak areas were compared with their respective standards to inase (Fig. 1b). Ann Microbiol (2019) 69:797–808 801 Fig. 1 Growth parameters, siderophores, and 1-aminocyclopropane-1- carboxylate (ACC) deaminase activity. a Effect of different bacterial iso- lates on root and shoot lengths of rice. b Amplification of acdS gene of SAK1 Fig. 2 Characteristics of bacterial isolates. a Quantification of ACC (1- aminocyclopropane-1-carboxylate) deaminase production. b Indole acetic acid (IAA) content observed in culture broth (CB) of SAK1. Quantification of ACC deaminase and phytohormone SAK1 was grown in culture broth (CB) with high NaCl concentrations production of SAK1 (100, 200, 300, and 400 mM). c Content of abscisic acid (ABA) in CB of SAK1. SAK1 was grown in CB with high NaCl concentrations (100, 200, 300, and 400 mM). Each data point is the mean of three replicates. Error SAK1 was evaluated for the production of ACC deaminase bars represent standard errors. The bars represented with different letters and phytohormones. We estimated the capability of endophyt- are significantly different from each other as evaluated by DMRTanalysis ic SAK1 bacteria to produce ACC deaminase on DF minimal media containing its substrate ACC. SAK1 revealed the highest value of deamination of ACC, up to 330 nmol α- ABA is produced in response to higher saline stress condition. −1 −1 ketobutyrate mg h (Fig. 2a). This also suggests that the production of ABA may be advan- Moreover, SAK1 produce ABA and IAA in LB media in tageous for the growth of certain species under salinity and the presence of different NaCl concentrations (0, 100, 200, drought stress conditions. Conversely, the decreased amount 300, and 400 mM). Here, we noticed that the amount of of IAA content was measured under higher salt concentration ABA was rising steadily with the increase in concentration in the broth, compared to that in normal culture media (Fig. of salt in the media (Fig. 2c). Thus, the increased amount of 2b). Hence, the production of ABA is augmented in the 802 Ann Microbiol (2019) 69:797–808 presence of higher NaCl concentration while the amount of acid were produced at concentrations of 3.3, 2.5, and −1 IAA is lowered down in the LB medium. 0.66 μgmL ,respectively. SAK1 mitigates salinity stress Bioactive GAs detected in culture filtrate of SAK1 Soybean plants inoculated with SAK1 revealed interesting The culture filtrate of isolated SAK1 was analyzed for the results under salinity stress conditions. The plants treated with purpose of GAs. Results of SAK1 analysis showed different SAK1 greatly mitigated the adverse effects of salinity stress, components of GAs, e.g., GA ,GA ,GA ,andGA .The 7 8 24 36 and favored plant growth, compared to untreated stressed −1 quantities of GAs were as follows: GA , 0.847 ng mL ;GA , 7 8 plants (Table 2). The SAK1-treated plants under salt stress −1 −1 0.04 ng mL ;GA , 0.024 ng mL ;and GA , 24 36 had significantly enhanced length and biomass compared to −1 0.038 ng mL , as shown in Fig. 3a. those of untreated plants. Under normal conditions (Table 2), soybean plants inoculated with the SAK1 had greatly en- Detection of organic acids in SAK1 hanced root/shoot length and weight and chlorophyll content compared to those of control plants. Our results also revealed Results of organic acids analysis showed that the cultured that the growth of soybean was found to decrease when the salt concentration was increased, but SAK1 inoculation miti- filtrate of SAK1 was containing butyric acid, succinic acid, acetic acid, and quinic acid (Fig. 3b). SAK1 revealed the gated the negative effects of salt on soybean growth. This −1 showed that the beneficial effects of endophytic SAK1 inter- higher amount (8.2 μgmL ) of acetic acid compare to other organic acids. Malic acid was detected at a concentration of action mitigated salinity stress and promoted root/shoot −1 growth and chlorophyll content at different salt concentrations 3.9 μgmL , whereas quinic acid, succinic acid, and butyric compared to those in control plants. Endogenous ABA and JA content of soybean An increase in ABA levels was observed under different NaCl concentrations for the inoculated and non-inoculated soybean plants (Fig. 4a). However, the amount of endogenous ABA content under different salt concentrations is greatly reduced because of SAK1 inoculation. This demonstrates the stress- mitigating capability of endophytic SAK1. However, there was no significant difference in endogenous ABA levels be- tween control and unstressed SAK1-treated plants (Fig. 4a). Furthermore, our results revealed that higher salt stress en- hances endogenous JA content in soybean plants. However, SAK1 treatment lowered the amount of JA in treated plants, when the results were compared to those of controls (Fig. 4b). Effect of SAK1 on antioxidant activities and total protein content of soybean The protein level increases proportionally to the increase in salt concentration in soybean (Fig. 5a). However, plants treat- ed with SAK1 showed a reduced amount of total protein at different salt concentrations. The application of SAK1 en- hanced total protein content in stressed conditions, compared to control plants (Fig. 5a). It has already been shown that reactive oxygen species Fig. 3 a Gas chromatography-mass spectrometry (GC-MS-SIM) analysis (ROS) are produced during elevated NaCl concentrations that and quantification of different gibberellins (GAs) by comparing them leads to oxidative stress of plants (Habib et al. 2016). with the internal standard. b Organic acids (low molecular weight) pro- Consequently, we also inspected the antioxidant enzyme ac- duced by SAK1. HPLC analysis and detection of organic acids used, in tivities in the present study (Fig. 5). Our results revealed that relation to their respective standards. Given letter(s) are significantly dif- ferent at the 5% level by DMRT salt stress significantly increased the activity of PPO in Ann Microbiol (2019) 69:797–808 803 Table 2 Effect of bacterial isolates on growth attributes of the plant exposed to different concentrations of salts Treatments SL (cm) RL (cm) SFW (g) RFW (g) CC (SPAD) Control 19.52 ± 2.2ab 16.94 ± 0.8abc 2.11 ± 0.3b 2.17 ± 0.4b 31.83 ± 0.9ab Isolate SAK1 22.04 ± 2.3a 18.66 ± 0.5a 2.27 ± 0.1a 2.60 ± 0.3a 33.03 ± 1.5a 100 mM NaCl 17.61 ± 2.0bc 16.50 ± 0.5bc 1.92 ± 0.3c 1.94 ± 0.4cb 30.69 ± 1.1bc 100 mM NaCl + SAK1 19.15 ± 2.1ab 18.13 ± 1.4ab 2.09 ± 0.4b 2.15 ± 0.3b 32.03 ± 1.0ab 200 mM NaCl 15.03 ± 4.1 cd 14.83 ± 0.9 dc 1.77 ± 0.3d 1.73 ± 0.5 cd 29.06 ± 1.2c 200 mM NaCl + SAK1 17.84 ± 2.9bc 16.36 ± 1.0bc 1.95 ± 0.5c 1.95 ± 0.4cb 31.46 ± 1.7ab 300 mM NaCl 12.61 ± 2.3d 12.86 ± 1.5d 0.84 ± 0.2f 1.40 ± 0.4e 27.96 ± 1.4d 300 mM NaCl + SAK1 15.60 ± 0.5bcd 13.36 ± 1.6d 1.17 ± 0.4e 1.46 ± 0.5de 28.50 ± 1.5c uninoculated plants, compared to that in SAK1-treated plants of POD in soybean plants under salt stress, compared to that in (Fig. 5b). Furthermore, SAK1 greatly reduced PPO activity in uninoculated plants (Fig. 5c). Contrary to this, the initial ap- soybean plants treated with different salt concentrations (100 plication of SAK1 induced POD activity to a large extent to 300 mM). However, no notable difference was observed compared to that in control unstressed plants (Fig. 5c). between inoculated and non-inoculated control plants (Fig. Reduced glutathione is a central cellular antioxidant and 5b). signaling compound that stimulates many vital cellular pro- As shown in Fig. 6c, the enzymatic activity of POD was cesses. Exposure to different concentration of salt stress enhanced in the plants, similar to that of total protein content, caused an increased formation of ROS and leads to oxidative under high salinity stress. SAK1 greatly arrested the activity stress. Our results demonstrated that glutathione concentra- tions were slightly higher in SAK1-inoculated soybean plants under salinity stress (Fig. 5d). In addition, a decrease in glu- tathione concentration was observed in soybean at all NaCl concentrations tested (100, 200, and 300 mM). It appears that no notable differences were observed in glutathione concen- trations between SAK1-inoculated and control soybean plants (Fig. 5d). The increase in glutathione content implicates a response to oxidative stress, to enhance plant growth and ame- liorate salt stress. Identification and phylogenetic analysis of SAK1 Molecular identification and phylogenetic analysis of SAK1 were performed by amplifying and sequencing the 16S rRNA gene and comparing it to a database of known 16S rRNA sequences. The results revealed that SAK1 exhibited a high level of 16S rRNA sequence identity (99.93%) with Curtobacterium oceanosedimentum ATCC31317 (T:superscript) (Fig. 6). The sequence was submitted to NCBI with the accession number MF949056. Discussion SAK1 was employed in the present study as salinity stress Fig. 4 Jasmonic acid (JA) and abscisic acid (ABA) content quantification relevant to enhance the plant tolerance against high salt in soybean. a Effect of SAK1 on the ABA content in soybean growing in high NaCl concentrations (100, 200, and 300 mM). b Effect of SAK1 on concentration. Biochemical analysis of SAK1 showed the the content of JA in soybean growing in high NaCl concentrations (100, bacterial strain produced different phytohormones such as 200, and 300 mM). Each data point is the mean of three replicates. Error ABA, JA, IAA, GAs, and antioxidants including polyphe- bars represent standard errors. The columns represented with different nolic oxidase, peroxidase, and reduced glutathione. The letters are significantly different from each other as evaluated by DMRT analysis SAK1 also produced different organic acids and ACC 804 Ann Microbiol (2019) 69:797–808 Fig. 5 Assessment of peroxidase, polyphenol oxidase, and total proteins in soybean. Effect of SAK1 on a total protein content, b polyphenol oxidase (PPO), c peroxidase, and d reduced glutathione activities in plants exposed to high concentrations of NaCl (SS1 = salt stress 1, SS2 = salt stress 2, SS3 = salt stress 3). Data are expressed as the mean of three replicates. Error bars represent standard errors, with different letters indicating statistically significant differences deaminase enzyme. Phytohormones, e.g., IAA and GAs, root and shoot growth. Bacterial ACC deaminase de- have been known growth hormones which enhance shoot grades ACC and reduces the deleterious effect of ethyl- length, root length, and number of root tips, leading to ene, ameliorating plant stress and promoting plant growth improve nutrients uptake and hence improve plant health (Cheng et al. 2007). Inoculation bacteria like SAK1 pro- under stress conditions (Ullah et al. 2013). ABA and JA ducing ACC deaminase induce longer roots which might are common stress response phytohormones which de- be helpful in the uptake of relatively more water soil un- grade the reactive oxygen species and protect plants from der drought/salinity stress conditions, thus increasing wa- the hazardous effects of stressors. In addition, antioxi- ter use efficiency of the plants under salinity (Shaharoona dants produced by SAK1 are linked with oxidative stress et al. 2006). tolerance. Ethylene is a gaseous plant growth regulator PGPB that produce ACC deaminase and phytohor- which regulates plant homoeostasis and results in reduced mones belong to various bacterial genera, including Fig. 6 Phylogenetic tree of SAK1 was constructed 16S rRNA sequences using neighbor joining (NJ) and maximum likelihood methods. Numbers above the branches represent the bootstrap values. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree Ann Microbiol (2019) 69:797–808 805 Arthrobacter, Microbacterium, Bacillus, Burkholderia, cellular toxicity in plants (Hasegawa et al. 2000; Mittova Rhizobium, Klebsiella,and Pseudomonas, that are known et al. 2004). To counteract adverse effects of ROS and oxida- to improve crop growth and enhance plant tolerance to tive stress, plants activate their antioxidant defense machinery, various abiotic and biotic stresses (Ali et al. 2018;Ali such as superoxide dismutase (SOD), POD, catalase (CAT), and Kim 2018; Egamberdieva et al. 2015; Gamalero and glutathione reductase (GR), that protect the plant against et al. 2010; Grover et al. 2011;Yue et al. 2007) including cellular stress and scavenge excess ROS (Kim et al. 2005). In salinity. Plant ACC is a main component of ethylene bio- the present study, SAK1-inoculated soybean plants showed synthesis which is a stress hormone produced during lower ROS levels, and the antioxidant enzyme activities (TP, stress and damages the plant tissues. The strain SAK1 PPO, and POD) were considerably reduced compared to those ACC deaminase degrades ACC and reduces the deleteri- of control soybean plants growing under different salinity ous effect of ethylene, ameliorating plant stress and pro- levels. Similar results were reported for lettuce, potato, and moting plant growth (Sergeeva et al. 2006). okra, where PGPB decreased the activities of ROS under in- Previous studies reported that salinity stress affects bio- creasing salinity stress (Gururani et al. 2013; Habib et al. chemical, morphological, and physiological functions in crop 2016; Hyo Shim Han 2005). plants (Shrivastava and Kumar 2015). Glutathione is an important antioxidant that regulates stress In this study, NaCl suppressed plant growth, and with in- responses by reacting with ROS and maintaining an intracel- creasing salt concentrations, a higher decline in plant growth lular redox state; it also enhances growth and salt tolerance in was observed (Table 2). However, with the help of SAK1, the plants (Chen et al. 2012; Csiszár et al. 2014). When intracel- adverse influence of NaCl stress on plant growth could be lular GSH concentrations are reduced, plants repeatedly dem- alleviated, by increasing biomass and chlorophyll content, onstrate a decrease in oxidative stress sensitivity (Grant et al. compared to control plants (Table 2). This clearly demon- 1997; Kushnir et al. 1995). In the present study, concentra- strates that SAK1 alleviates the debilitating effects of salt tions of glutathione were found to be slightly higher in SAK1- stress. Irizarry and White (2017) reported that inoculated soybean plants than in non-inoculated plants. It has Curtobacterium oceanosedimentum produce IAA and been previously reported that glutathione regulates biotic and enhanced the growth of cotton plant under salinity stress. abiotic stress responses, and plays a pivotal role in the photo- Similarly, Bianco and Defez (2009, 2010) reported that in synthetic regulation of plants (Diaz-Vivancos et al. 2015; Gill saline environment IAA-producing bacteria also increase root et al. 2013). In SAK1-inoculated soybean plants, increases in and shoot length of chickpea and Medicago plants, compare to glutathione levels were observed compared to those in non- uninoculated plants. inoculated plants, which indicated a comparatively high po- Abscisic acid (ABA), a phytohormone important for tential of ROS scavenging that increased photosynthesis, the growth and development of plants, plays a vital role growth, and shoot biomass. Our findings support the results in many stress signaling pathways, and is a notable plant of Fatma et al. (2014), who also found that an increase in stress marker. Plants adjust ABA levels constantly glutathione levels during salt stress could improve growth through stomatal closure, minimizing water loss and ac- and photosynthesis in plants. tivating many stress-responsive genes, in response to Together, our results lead to the conclusion that the appli- changing physiological and environmental conditions cation of Curtobacterium sp. SAK1 greatly mitigates the ef- (Zhang et al. 2006). Salinity stress leads to an increase fects of salt stress and promotes plant growth in G. max. in ABA content (Wang et al. 2001). Our findings show Authors’ contributions MAK, SA, SMK, and SA conducted the exper- that plants inoculated with SAK1 significantly produce iments. ALK and IU helped in writing of the manuscript. IJL designed, lowers endogenous ABA production compared to non- supervised, and financed the research. All authors have read and agreed to inoculated plants (Fig. 4a). The production of ABA by its content and also that the manuscript conforms to the journal’spolicies. PGPB improves growth of the plant and mitigates salt stress (Cohen et al. 2008). Suppressive influence of sa- Funding This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) linity stress on plant germination has been associated funded by the Ministry of Education (2016R1A6A1A05011910). with a reduced level of endogenous hormones. Endophytic originated plant growth regulators have been Compliance with ethical standards found to have same effects in plant as of exogenous phytohormones (Ullah et al. 2019). In this regard, the Conflicts of interest The authors declare that they have no conflict of SAK1, used in present study, could be a new addition interest. in batch of plant stress hormone-regulating endophytic bacteria (PSHEB). 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Published: Apr 12, 2019

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