J. Microbiol. Biotechnol. 2020. 30(5): 717–725 https://doi.org/10.4014/jmb.1911.11063 Inoculation with Indole-3-Acetic Acid- Producing Rhizospheric Rhodobacter sphaeroides KE149 Augments Growth of Adzuki Bean Plants Under Water Stress Sang-Mo Kang1, Arjun Adhikari1, Ko-Eun Lee1, Muhammad Aaqil Khan1, Abdul Latif Khan2, Raheem Shahzad3,4, Sanjeev Kumar Dhungana5, and In-Jung Lee1* 1School of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea 2Natural and Medical Science Research Center, University of Nizwa, Nizwa 616, Oman 3Department of Biology, College of Science, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Damam 31441, Saudi Arabia 4Basic and Applied Scientific Research Center, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Damam 31441, Saudi Arabia 5Department of Southern Area Crop Science, National Institute of Crop Science Rural Development Administration, Miryang 50424, Republic of Korea The use of plant growth-promoting rhizobacteria is economically viable and environmentally safe for mitigating various plant stresses. Abiotic stresses such as flood and drought are a serious threat to modern agriculture. In the present study, the indole-3-acetic acid-producing rhizobacterium R. sphaeroides KE149 was selected, and its effects on the growth of adzuki bean plants under flood stress (FS) and drought stress (DS) were investigated. IAA quantification of bacterial pure culture revealed that KE149 produced a significant amount of IAA. Moreover, KE149 inoculation notably decreased stress-responsive endogenous abscisic acid and jasmonic acid and increased salicylic acid in plants under DS and FS. KE149 inoculation also increased proline under DS and methionine under FS. In addition, KE149 inoculation significantly increased the levels of calcium (Ca), magnesium (Mg), and potassium (K) while lowering the sodium (Na) content in the plant shoot under stress. KE149-treated plants had markedly greater root length, shoot length, stem diameter, biomass, and higher chlorophyll content under both normal and stressed conditions. These results suggest that KE149 could be an efficient biofertilizer for mitigating water stress. Keywords: Abiotic stress, drought, flood, phytohormones, Rhodobacter sphaeroides KE149 Received: November 27, 2019 Accepted: February 9, 2020 Introduction First published online: The adzuki bean plant is a leguminous plant consumed as folk medicine and is one of the important crops February 10, 2020 especially in countries such as China, Korea, and Japan [1]. It has rich antioxidant properties and medicinal importance [2]. However, studies on adzuki bean cultivation are limited [3]. Abiotic factors such as flood, *Corresponding author Phone: +82-53-950-5708 drought, salinity, extreme temperature, and nutrient unavailability are considered as the major causes for a Fax: +82-53-953-697 reduction of almost 50% of the potential crop yield [4]. Drought stress has led to crop production damage (> 7%), E-mail: [email protected] which is 8–11% greater in developed countries compared with developing countries globally [5]. Despite various scientific approaches, the amelioration of drought stress and flood stress is still challenging for quality crop Supplementary data for this production. Accordingly, a better understanding of the physiology beyond flood stress and drought stress and its paper are available on-line only biological remediation could broaden the scope of research. at http://jmb.or.kr. In order to adapt to stress, plants attempt to morphologically change their intercellular formation through the induction of several signaling regulators such as plant hormones and ROS [6]. Among the various pISSN 1017-7825 phytohormones, indole-3-acetic acid (IAA), salicylic acid (SA), abscisic acid (ABA), and jasmonic acid (JA) play eISSN 1738-8872 an important role in signaling and cross-communication. Under flood stress, plants are unable to absorb oxygen Copyright© 2020 by for normal physiological activities because they cannot generate glucose, causing several metabolic problems. The Korean Society for During drought, plants tend to accumulate ABA, and auxin levels are usually decreased [7]. Microbiology and A mechanism that has been identified in rice under drought is that JA can modulate root hydraulic conductivity Biotechnology to enhance drought tolerance. Specifically, the rice bHLH protein (OsbHLH148) interacts with OSJAZ1 (jasmonic acid-regulated gene) and activates OsDREB1 (a protein for drought tolerance), enhancing drought stress May 2020⎪ Vol. 30 ⎪ No. 5 718 Kang et al. tolerance. In addition, the biosynthesis and transport of ABA are mediated by ABA signaling receptors (2C-type protein phosphatases), which transport ions to the stomata, resulting in the influx of CO2 and efflux of H2O and leading to stomatal closure [8]. SA biosynthesis requires the primary metabolite chorismate, which involves isochorismate synthase (ICS) and phenylalanine ammonia lyase (PAL) [9]. Moreover, under drought, AUX/IAA is conjugated to amino acids, which is catalyzed by GH3 proteins. IAA/AUX is localized in cells and degraded when it interacts with auxin-signaling F-Box protein. After degradation, auxin response factors are released, which mediate the transcription of auxin-responsive genes [8]. In recent years, plant growth-promoting rhizobacteria (PGPR) have been reported to mitigate plant stress and promote growth through phytohormone modulation, antioxidant and amino acid regulation, and nutrient assimilation. These microorganisms colonize the root rhizosphere and acquire several micronutrients and macronutrients such as calcium (Ca), magnesium (Mg), potassium (K), and phosphorus (P) from the soil. These nutrients play a key role in regulating the amino acid pathway. Amino acids are considered as precursors for the defense system in plants, and they play an important role in the cross talk between SA and JA, affecting the plant- microbe interaction [10, 11]. Amino acids are osmotically active substances that contribute to osmotic pressure adjustments during water stress. Amino acids maintain the negative osmotic potential of cells during drought stress, thus keeping the cell wall rigid and preventing cells from shrinking due to dehydration [12]. Under flood stress, methionine has been reported to act as a precursor in the synthesis of ethylene, which is produced through 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC synthase. During ethylene biosynthesis, ACC is converted to ethylene, and oxygen is vital for the production of ethylene. The absence of oxygen in the root zone during flood may disrupt plant growth [7]. The application of PGPR could regulate plant metabolism by enhancing methionine production and promoting plant growth. These effects have been reported for several microorganisms such as Pseudomonas putida [13], Promicromonospora sp. SE188, Burkholderia cepacia SE4, and Acinetobacter calcoaceticus SE370 [14]. In our previous study, Rhodobacter sphaeroides was found to promote the growth of cucumber plants by producing IAA [15]. Our current analysis further revealed the innate ability of the R. sphaeroides strain to produce IAA. [16]. IAA producing bacteria effectively protect plants against various biotic and abiotic stresses and can assist plant growth [17]. Rhizobacteria use tryptophan in root exudates and convert them into IAA, which is utilized by the plant roots and hence activate the plants auxin signaling pathway, which ultimately led to proliferation and growth promotion of plant cells [18]. Therefore, we hypothesized that the application of IAA- synthesizing R. sphaeroides could confer resistance against flood stress and drought stress in soybean plants. Materials and Methods Bacterial Inoculum Preparation The bacterial strain was grown in 250 ml of LB broth (tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0 ± 0.2; autoclaved for 15 min at 121°C) for 5 days and centrifuged at 5,000 ×g for 15 min to separate the culture broth and bacterial cells. The cells were separated, diluted with sterilized distilled water and used for inoculation in plants. IAA Detection and Quantification The method described by Lee et al. [19] was followed to detect and quantify the IAA production in KE149 culture. The details are mentioned in supplementary file. Extraction and Quantification of ABA in Bacterial Pure Culture ABA was extracted and quantified using the method described by Shahzad et al. [20]. In brief, R. sphaeroides was grown in LB media (tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0 ± 0.2; autoclaved for 15 min at 121°C) for 3 and 5 days. Then, 25 ml of broth was taken, and the pH was adjusted to 2.5. The extract was obtained using ethyl acetate. The pure culture filtrate in which R. sphaeroides KE149 was grown was supplemented with [(±)- 3,5,5,7,7,7-d6]-ABA as an internal standard. Polyvinylpolypyrrolidone was added, and the mixture was stirred at 120 rpm for 40 min, followed by filtration; the pH was adjusted to 3.5, and the mixture was partitioned by EtOAc. The extracts were left to evaporate, and ABA was obtained using diethyl ether/methanol (3:2, v/v) and dichloromethane. The purified ABA extracts were dried, methylated, and analyzed by GC-MS/SIM (6890N Network GC System and 5973 Network Mass Selective Detector; Agilent Technologies, USA). The Lab-Base (ThermoQuset, UK) data system software was used to monitor the signal ions at m/z 162 and 190 for Me-ABA and 2 m/z 166 and 194 for Me-[ H6]-ABA. The experiment was repeated three times. Plant