Spore associated bacteria regulates maize root K+/Na+ ion homeostasis to promote salinity tolerance during arbuscular mycorrhizal symbiosis

Spore associated bacteria regulates maize root K+/Na+ ion homeostasis to promote salinity... Background: The interaction between arbuscular mycorrhizal fungi (AMF) and AMF spore associated bacteria (SAB) were previously found to improve mycorrhizal symbiotic efficiency under saline stress, however, the information about the molecular basis of this interaction remain unknown. Therefore, the present study aimed to investigate the response of maize plants to co-inoculation of AMF and SAB under salinity stress. Results: The co-inoculation of AMF and SAB significantly improved plant dry weight, nutrient content of shoot and root tissues under 25 or 50 mM NaCl. Importantly, co-inoculation significantly reduced the accumulation of proline + + + in shoots and Na in roots. Co-inoculated maize plants also exhibited high K /Na ratios in roots at 25 mM NaCl concentration. Mycorrhizal colonization significantly positively altered the expression of ZmAKT2, ZmSOS1, and + + ZmSKOR genes, to maintain K and Na ion homeostasis. Confocal laser scanning microscope (CLSM) view showed that SAB were able to move and localize into inter- and intracellular spaces of maize roots and were closely associated with the spore outer hyaline layer. Conclusion: These new findings indicate that co-inoculation of AMF and SAB effectively alleviates the detrimental + + effects of salinity through regulation of SOS pathway gene expression and K /Na homeostasis to improve maize plant growth. Keywords: Arbuscular mycorrhizal fungi, Spore associated bacteria, Plant-microbe interaction, gfp-tagging, Endophytic localization, Salt stress Background Cl ions are taken up by the plant cells causing toxic effects The salinity of soil is one of the most important concerns, such as damage to cell organelles and plasma membrane, which are increasing progressively worldwide. More than disruption of cell organelles, photosynthesis, and protein 800 million hectares (over 6%) of the world’s total land synthesis [4, 5]. As the majority of crop plants are glyco- area are affected by soil salinity (FAO 2005). Increasing phytes, their tolerance to salinity level beyond the threshold salinization of arable lands adversely affects crop estab- level reduces productivity [6]. Maize is the third most lishment, growth, and development contributing to important cereal crop in the world especially in developing huge losses in productivity [1, 2]. The high concentra- countries [7] and is considered as a salt sensitive cereal tion of salt present in the soil causes both hyper-ionic crop [8, 9]. In maize, Na is a major ion and under salt and hyper-osmotic stress and leads to plant death [3]. stress, it causes ion toxicity in plants [10]. Under prolonged salinity stress, the excessive Na and The interaction between plant roots and salt-tolerant microorganisms helps plants alleviate the deleterious effects * Correspondence: tomsa@chungbuk.ac.kr of salinity. Arbuscular mycorrhizal fungi (AMF) can form a Department of Environmental and Biological Chemistry, College of mutualistic association with the roots of more than 80% of Agriculture, Life and Environment Sciences, Chungbuk National University, the terrestrial plants [11]. AMF have been reported to Cheongju, Chungbuk 361-763, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 2 of 13 enhance plant growth under different salinity levels mechanism involved in the alleviation of salinity stress in [5, 12–14] by enhancing the nutrient acquisition in plants. host plants. The alleviation of salt stress by AMF has been Therefore, this study aimed to investigate the effects of reported through increased osmotic balance, increased the co-inoculation of AMF and SAB on the growth of activity of anti-oxidant enzymes, increased photosynthetic maize plant under salinity stress. The study also evalu- activity [15], increased levels of osmoregulant (proline) ated the association of SAB with AMF spore walls and [16] and enhanced water uptake in plants [17]. In addition localization in plant roots; and analyzed the alteration in to the plant, AMF also interacts with many bacterial the expressions of genes involved in ion homeostasis by species in a natural environment. The interactions between AMF and SAB under salinity stress. AMF and bacteria have been shown to improve mutualistic fungus-host interaction [18]and plantgrowth [19]. Some Methods studies have reported the positive effects of co-inoculation Strains detail of AMF and plant growth promoting (PGP) bacteria on Pseudomonas koreensis S2CB35, a SAB, was isolated from plant growth and nutrient uptake under saline stress con- the spore walls of AMF (Gigasporaceae) and demonstrated ditions [20, 21]. Furthermore, many soil microorganisms spore association characteristics as described earlier [35]. and plant endophytic bacteria have been studied and The isolated bacterial strain exhibited multiple plant reported to promote plant growth under various environ- growth-promoting characteristics, such as reduced ethylene mental conditions [22–25]. In our recent study [26], we stress and improved early growth of maize under salt stress found that AMF spore associated bacteria significantly [26]. Two AMF strains, Gigaspora margarita S–23 and reduced ethylene stress level and improved maize seedling Claroideoglomus lamellosum S–11, were used in the growth. Co-inoculation of AMF and mycorrhizosphere present study. They were isolated from a salt affected bacteria increased maize plant growth by enhancing AMF coastal reclamation land of Saemangeum in South hyphal length and facilitating P uptake [27]. However, the Korea and propagated for the mass multiplications by a define mechanisms by which the microbes alleviate salt single spore mass production technique [36]. The detail stress in plants remain unclear. of the strains and protein maker used in this study are Due to the similar physiochemical structure of Na giveninTable 1. + + and K , under salt stress, the excess of Na osmoticum competes for K entry into the symplast, at the transport Green fluorescent protein (gfp)-tagging of the bacterial + + sites. The large cytosolic Na ions compete for K binding strain sites and crucially restricts the metabolic activities that In order to monitor the activity of SAB, the strain was + + require K . The K ion is a key component in the cytosol tagged with GFP before inoculation. The insertion of the as it plays a critical role in protein synthesis, activation of mini-Tn5 gusA::gfp cassette into pB10 was performed by enzymes and photosynthesis, turgor maintenance and introducing Escherichia coli gene Tn5 gusA gfp cassette stomatal movement [28]. AMF is known to selectively (pFAJ1820) [37] into strain P. koreensis S2CB35 by + 2+ uptake K and Ca , which act as osmotic equivalents triparental mating with the helper plasmid pRK2013 of as they avoid the uptake of toxic Na [29]. However, E. coli HB101. The transformants were selected on a the molecular mechanisms of regulation of uptake of K half-strength nutrient agar medium supplemented with + − 1 and exclusion of Na in plants by microbial inoculation kanamycin at 50 μgmL . The presence of GFP in the remain to be elucidated. The salt overly sensitive (SOS) purified transformants was confirmed by PCR amplifica- signaling pathway plays a significant role in maintaining tion using following primers: YL065 (F) 5’ GCGATGTTA + + ion homeostasis by regulating Na and K transport at the ATGGGCAAAAA-3′ and YL066 (R) 5’-TCCATGCCA plasma membrane and tonoplast. The key genes respon- TGTGTAATCCT-3′. The thermal cycling program for sible for ion homeostasis are SOS1, SKOR,and AKT2 [30]. the amplification consisted of initial denaturation at 94 °C Among these, SOS1 is widely studied for its ability to for 3 min, followed by 35 cycles at 94 °C for 30 s, extrude Na and control xylem loading for a long-distance annealing at 56 °C for 1 min, and extension at 72 °C for Na transport [31, 32]. SKOR is involved in the transloca- 1 min, with the final extension at 72 °C for 10 min [38]. tion of K toward shoots through xylem [33]. Further, the The resulting amplicon of 650-bp was confirmed by gel phloem expressing K channel, AKT2, is also involved in electrophoresis. The relative fluorescence activity of the translocation of K in shoots [34]. A previous study gfp-mutant derivatives was analyzed using a flow cytometer conducted by Estrada et al. (2013) reported that these (FACScalibur) equipped with an air-cooled argon-ion laser genes are differentially regulated by AMF regulating ion emitting at 488 nm (15 mW) [39]. Single gfp-derivative homeostasis in plants under salt stress. Moreover, eluci- of P. koreensis S2CB35 was differentiated based on cell dating the gene expression regulated by endophytic morphology, colony appearance, and growth rate char- bacteria might provide broad insights into the molecular acteristics of the wild type. Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 3 of 13 Table 1 Bacterial strains and plasmids used in the present study Plasmid/strain Genotype or other relevant characteristics Reference Escherichia coli S17–1 Vector for plasmid pFAJ1820 Xi et al. [37] Escherichia coli HB101 Helper bacteria containing pRK2013 plasmid Xi et al. [37] P. koreensis S2CB35 PGP-SAB (Genbank number: KM507143) Selvakumar et al. [26] P. koreensis S2CB35-gfp gfp-tagged mutant representative of P. koreensis S2CB35 This study Gigaspora margarita S-23 Propagated AMF (Genbank number: KP677599) Selvakumar et al. [36] Claroideoglomus lamellosum S-11 Propagated AMF (Genbank number: KP677595) Selvakumar et al. [36] pRK2013 Mobilizing plasmid Figurski & Helinski [74] pFAJ1820 pUT mini Tn5gusA-pgfp Xi et al. [37] Soil analysis and seedling preparation which was obtained from 75 g of non-autoclaved AMF The soil sample was collected from low salt affected inoculum, whereas G. margarita inoculated pots received reclamation land of Saemangeum, South Korea. The extracts of C. lamellosum and vice versa. Equally grown physicochemical properties of soil were analyzed using 7-day-old maize seedlings were transplanted into pots the standard laboratory protocols. The pH of soil was 6.0, containing 2.5 kg of soil and maintained for 44 days after with electrical conductivity (EC) value of 0.34 dS/m, organic transplantation (DAT). Each plant was supplemented with matter of 5.5 g/kg, available phosphorus of 40.66 mg/kg, 100 mL of modified Hoagland’s nutrient solution [40] and with content of K, Ca, Mg, and Na measured as 0.56, regularly. 1.0, 2.2, and 0.79 cmolc/kg, respectively. The soil texture For molecular gene expression analysis and confocal was sand 76%, silt 23.2% and clay 0.8%. The maize laser scanning microscopy (CLSM), a separate set was seeds (Zea mays L.) were surface sterilized using 70% prepared with the same treatments. The microbial inocu- ethanol for 1 min, treated for 5 min with 6% NaOCl, lation was applied in the same ratio in 600 mL pots and washed seven times with sterile distilled water. For containing the same soil with an exception that the soil the bacterial treatment, the surface-sterilized seeds were was autoclaved for 3 days consecutively to destroy all imbibed in 10 mL of 0.1 M phosphate buffer (pH 6.8) the microbes present in the soil. Both the experiments were containing 1 × 10 cfu/mL of P. koreensis (S2CB35) for conductedsimultaneouslyunder thesameenvironmental 4 h before the seeds were sown in a seedling tray. For conditions. the control and AMF-alone treatments, the seeds were For seed bacterization, the maize seeds were soaked in treated with 0.1 M phosphate buffer (pH 6.8) with sterile 0.1 M phosphate buffer (pH 6.8) containing 1 × 10 cfu/mL bacteria. of SAB for 2 to 4 h. In addition to seed bacterization, 5 mL of 0.1 M phosphate buffer (pH 6.8) containing Inoculation treatments and salt stress conditions 1×10 cfu/mL of SAB was added to the SAB and In order to study the effects of AMF and SAB on maize co-inoculation treatment pots at 10 and 30 DAT. The growth under NaCl stress, we designed six treatments control and AMF-alone treatments received the same groups designated as T1 for non-treatment control and amount of bacterial culture with the exception that it T2, T3, T4, T5, and T6 for treatments with G. margarita, was autoclaved at 121 °C for 15 min before inoculation. C. lamellosum, SAB, G. margarita + SAB and C. lamello- Salt stress was produced with three different NaCl sum + SAB, respectively, and each one irrigated with three concentrations (0 mM, 25 mM, and 50 mM) at 23 different concentrations of NaCl (0 mM, 25 mM, and DAT. To avoid osmotic shock, NaCl stress was induced 50 mM). The pot experiment was performed in a com- gradually by adding 10 mM and 15 mM to each pot after pletely randomized block design with four replications. every alternate day, and the desired salt concentration was Each pot was filled with 2.5 kg of soil, and the mycorrhizal achieved after 5 days. Leaching of water from the pots was treatment pots received 75 g (3%) of AMF inoculum (each prevented by maintaining the soil water to a level below AMF inoculum containing approximately 200 spores the field capacity at all the times. The maize plants were and 30 root bits), which was added 1 cm below the soil grown for another 15 days under the salt stress condition surface. The control and SAB treatments received 75 g and then harvested. The soil EC value measured at the of autoclaved AMF inoculum to maintain the same nutrient time of harvest for NaCl stress of 0 mM, 25 mM, and content. In addition, to maintain the similar bacterial popu- 50 mM were 0.49 ± 0.09 dS/m, 2.52 ± 0.35 dS/m, and lation in all of the treatments, control, and SAB treatments 4.50 ± 0.12 dS/m, respectively. At the end of the experiment, received 70 mL of soil extract of each AMF inoculum, the plants were harvested carefully, washed in distilled water, Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 4 of 13 separated into leaves, shoots, and roots and were used for GTCCACTTCACTACAC-3′). cDNAs that originated the analysis of different parameters. from three different biological samples were used for each gene analysis. Alpha tubulin (gi:450292) and polyubiquitin Determination of mineral nutrients (gi:248338) were used as the internal controls for the The biomass or dry weight of the shoots was determined normalization of data. All experiments were done in tripli- after oven drying at 70 °C for at least 48 h. The proline cate with three repeats. content of the leaf was estimated according to the method described by Bates et al. [41]. The total nitrogen Preparation of plant root and spore samples for confocal accumulation in the plants was measured using a Kjeldahl microscopy analyzer (K9860 Kjeldahl Analyzer, Hanon Instruments). For confocal scanning laser microscopy (CLSM), the The available phosphorus was determined using the root and soil samples were used from the 600 mL pot vanadate-molybdate method, and the Ca, Mg, Na and K experiment. The fresh root samples removed from SAB concentrations were estimated using inductively coupled treated plant were washed in sterile distilled water and plasma optical emission spectrometry (ICP-OES). dried on a blotting paper. The roots were surface-steril- Chloride anions were determined in an aqueous ized and aseptically sectioned with sterile scalpel blades. extraction obtained from 0.4 g of dry plant material using The sections were mounted on a slide using fluores- 20 mL of deionized water. The extract was shaken for 2 h cence mounting medium under a coverslip. For the and then filtered through a Whatman number 2 filter spores, the isolated spores were either mounted directly paper and a 0.45 μm nylon membrane filter (Millipore). or after surface sterilization with 2% chloramine T and The diluted filtrate was then injected into an ion-exchange 100 μg/mL streptomycin for 30 min. The microscopic ob- chromatography system (Metrohm) packed with anion servation of root and spore samples were performed using separation column Metrosep A Supp 5. a Leica TCS SP2 confocal system (Leica Microsystems Hei- delberg GmbH) equipped with an Ar laser (gfp: Mycorrhizal development excitation, 488 nm; emission filter BP, 500 to 530). Image The root samples were washed with tap water to remove acquisitions were performed under the objectives 20× and the adhering soils, and the roots were cut into the pieces of 40× (N.A. approximately 0.75) and were processed using 1 cm length and stained with 0.05% trypan blue as per the the Zen lite 2012 (blue edition). method described by Phillips & Hayman [42]. The mycor- rhizal root colonization (M%), colonization frequency (F%) Statistical analysis and arbuscules abundances in the whole root system (A%) The data were statistically analyzed using analysis of were calculated according to Trouvelot et al. [43]. The variance (ANOVA) for a completely randomized block isolation of AMF spores from 50 g of soil was carried design with SAS package 9.4 software and the differences out by wet sieving and decanting method [44]. in means were determined by the least significant differ- ences (LSD). Duncan’s multiple-range test was performed Quantitative real time PCR at P ≤ 0.05 on each of the significant variables measured. The maize root samples collected from the 600 mL pot P values less than 0.05 were considered as statistically experiment plants were washed under running water significant. and then rinsed three times with distilled water and frozen in liquid nitrogen, ground and stored at − 80 °C. Total Results RNA was extracted using the RNeasy plant mini kit Plant growth, proline content and mycorrhizal (Qiagen, Valencia, CA, USA) from the root samples stored parameters at − 80 °C. cDNA was synthesized using Superscript III first Microbial inoculation effect on maize plant growth was strand synthesis system (Invitrogen). The gene expression assessed. A significantly negative effect of salinity on analyses were carried out by quantitative reverse transcrip- growth of the plant was observed in all the treatments tion (qRT)-PCR using CFX96 Real-Time System (Bio-Rad and a more prominent effect was evident at the highest Laboratories, München, Germany) with SYBR Green salt concentration of 50 mM NaCl. Treatments with master mix (iQ SYBR Green Supermix, Bio-Rad). Specific AMF and SAB significantly increased the dry weight of primers were designed by Estrada et al. [9] and used to the maize as compared to the control in all salt concen- analyze the genes: ZmAKT2,For (5′-CCTCAAGCATC tration (Fig. 1a). Among the microbial treatments, co- AGGTCGAGA-3′)and ZmAKT2, Rev. (5′-CTCTGTAAT inoculation of C. lamellosum with SAB significantly CTTCCTGGACG-3′), ZmSKOR, For (5′-TCAGATCCA increased plant dry weight at 25 and 50 mM NaCl. Further, AGATGTCCCAG-3′)and ZmSKOR, Rev. (5′-TTCGTA with increasing salt concentration, the corresponding TCCTCTTAACGCAG-3′), ZmSOS1, For (5′-GCTTGTC increase in proline content was observed in all the treat- ACATACTTCACAG-3′)and ZmSOS1,Rev.(5′-ACTT ments (Fig. 1b). However, at 0 mM NaCl, no significant Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 5 of 13 ac bd Fig. 1 AMF and SAB co-inoculation effect on plant growth and mycorrhizal development. a Plant dry weight, b Leaf proline content, c AMF spore count d Mycorrhizal colonization. T1 – control, T2 - Gigaspora margarita S-23, T3 – Claroideoglomus lamellosum S-11, T4 – Pseudomonas koreensis S2CB35, T5 – T2 + T4, T6 – T3 + T4. Plants were subjected to 0 (0.5 dS/m), 25 (2.5 dS/m) or 50 mM NaCl (4.5 dS/m). Different letters indicate significant differences (P < 0.05) among the treatments at each salt level (a, b, c, d, e, f) or among salt levels for each treatment: T1 (A, B, C), T2 (D, E, F), T3 (G, H, I), T4 (J, K, L), T5 (M, N, O) or T6 (P, Q, R). Each value represents the mean of four replicates ± standard error (SE) differences in leaf proline content were observed between uptake in both shoot and root of maize was estimated treatments and control. At 25 mM NaCl, co-inoculation with microbial treatments in all salt levels (Tables 2 with AMF and SAB or C. lamellosum alone treatment and 3). Single and co-inoculation of AMF and SAB significantly reduced the proline content. At 50 mM NaCl, significantly increased total-N and phosphorous in both co-inoculation of C. lamellosum with SAB and only SAB shoot and root at all salt levels. A significantly higher treatments significantly reduced the proline content. potassium uptake was observed for co-inoculation of The effect of salinity on mycorrhizal spore count is shown AMF and SAB in both shoot and root tissues; however, at in Fig. 1c. The increase in salinity reduced AMF spore 50 mM of NaCl, G. margarita with SAB co-inoculation in count. At 0 mM NaCl, the co-inoculation of AMF and shoot and C. lamellosum with SAB co-inoculation in root SAB significantly increased spore count than AMF alone were not significantly different from control. A significantly treatment. However, no significant differences were higher calcium accumulation in shoots was observed for observed between the AMF and AMF co-inoculation with AMF andSAB co-inoculatedplants at0and50mMNaCl. SAB treatments at 25 and 50 mM of NaCl concentration. In root, co-inoculation of AMF and SAB significantly Mycorrhizal colonization in maize roots was negatively improved calcium accumulation at 0 and 50 mM NaCl. affected by increasing salinity (Fig. 1d). A significantly Furthermore, co-inoculation treatment showed signifi- high mycorrhizal colonization was observed with co-in- cantly higher magnesium accumulation in shoots except at oculation of AMF and SAB in all salt concentrations 50 mM NaCl, whereas in roots, co-inoculation treatment compared to AMF alone treatment. Likewise, co-inoculation significantly improved magnesium accumulation at all salt of AMF and SAB exhibited significantly high colonization levels except for C. lamellosum with SAB co-inoculation frequency and arbuscules abundance than AMF alone treatment at 50 mM NaCl. treatment (Additional file 1: Figure S1). Sodium and chloride uptake Nutrient accumulation Under salt stress, plants take up more sodium ion than The efficiency of nutrient uptake by plants under salt potassium. A significant increase in sodium accumula- stress shows the degree of plant response to such stress. tion in maize shoots was observed with the increase in The highest salinity at 50 mM of NaCl concentration salinity (Fig. 2a). At 0 and 25 mM NaCl, no significant significantly lowered the nutrient uptake by plants in all difference was observed between the treatments and the treatments. However, a significantly increased nutrient control. However, at 50 mM NaCl, the co-inoculation of Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 6 of 13 Table 2 Effect of AMF and SAB co-inoculation on maize shoot nutrient accumulation under different salinity levels Salt concentration Treatments Total N P K Ca Mg mg / plant c, A c, A b, A c, A b, A 0 mM T1 819.6 ± 64.3 68.3 ± 6.8 470.4 ± 46.3 40.0 ± 4.9 32.9 ± 1.5 bc, D b, D b, D bc, DE b, DE T2 1368.4 ± 247.1 115.3 ± 11.2 544.5 ± 50.4 45.9 ± 7.0 41.4 ± 5.1 bc, G b, G b, G bc, H b, G T3 1362.9 ± 185.3 113.1 ± 9.9 499.4 ± 74.1 41.1 ± 6.5 42.6 ± 6.1 bc, J b, J ab, J bc, JK b, K T4 1342.7 ± 101.9 133.0 ± 16.9 597.2 ± 137.8 42.0 ± 11.9 36.9 ± 7.1 a, M a, M a, M b, M a, M T5 1997.7 ± 311.2 174.4 ± 15.4 838.0 ± 49.3 69.4 ± 5.7 64.3 ± 3.3 ab, P a, P a, P a, P a, P T6 1921.7 ± 68.9 180.1 ± 9.1 817.0 ± 57.6 96.3 ± 11.2 69.6 ± 2.2 c, AB d, A d, A b, A b, A 25 mM T1 808.3 ± 40.5 66.3 ± 1.1 425.2 ± 11.0 47.0 ± 5.7 46.2 ± 2.3 ab, D bc, D cd, D ab, D ab, D T2 1415.6 ± 96.6 101.8 ± 7.8 470.1 ± 63.5 63.3 ± 8.6 58.7 ± 8.2 b, G c, G bcd, G a, G ab, G T3 1218.6 ± 142.3 98.3 ± 3.2 566.4 ± 28.9 82.1 ± 13.8 66.9 ± 9.4 ab, J b, J abc, J ab, J ab, J T4 1441.1 ± 133.9 129.8 ± 10.5 617.8 ± 44.3 67.3 ± 7.3 66.8 ± 5.8 a, M b, N ab, N ab, M a, M T5 1729.2 ± 117.8 130.6 ± 5.7 697.1 ± 77.9 75.4 ± 5.3 71.0 ± 7.0 a, P a, P a, P a, P a, P T6 1765.1 ± 123.7 161.5 ± 16.5 757.1 ± 70.0 87.4 ± 10.1 78.4 ± 5.7 b, B d, B b, B b, A a, A 50 mM T1 553.4 ± 78.9 50.4 ± 2.2 295.4 ± 31.4 30.5 ± 3.9 42.0 ± 12.5 b, E c, E b, E b, E a, E T2 768.4 ± 24.3 67.8 ± 4.4 287.2 ± 23.3 31.1 ± 2.2 31.9 ± 1.8 b, H bc, H b, H b, H a, G T3 764.1 ± 71.2 72.6 ± 2.9 274.1 ± 18.8 30.2 ± 1.3 50.3 ± 17.8 b, K c, K b, K b, K a, K T4 734.2 ± 98.9 69.7 ± 5.7 304.6 ± 20.7 29.8 ± 4.2 33.2 ± 4.6 a, M b, O b, O a, N a, N T5 1066.9 ± 21.5 87.5 ± 4.1 352.3 ± 12.2 50.6 ± 3.7 46.1 ± 1.6 a, Q a, Q a, Q a, P a, P T6 1265.4 ± 170.4 110.5 ± 11.6 467.4 ± 40.8 63.4 ± 10.8 60.7 ± 8.6 T1 – control, T2 - Gigaspora margarita S-23, T3 – Claroideoglomus lamellosum S-11, T4 – Pseudomonas koreensis S2CB35, T5 – T2 + T4, T6 – T3 + T4. Plants were subjected to 0 (0.5 dS/m), 25 (2.5 dS/m) or 50 mM NaCl (4.5 dS/m). Different letters indicate significant differences (P < 0.05) among the treatments at each salt level (a, b, c, d, e, f) or among salt levels for each treatment: T1 (A, B, C), T2 (D, E, F), T3 (G, H, I), T4 (J, K, L), T5 (M, N, O) or T6 (P, Q, R). Each value represents the mean of four replicates ± standard error (SE) + + C. lamellosum with SAB significantly enhanced sodium K /Na ratios + + accumulation in a shoot. In root tissues, the sodium In both shoots and roots of maize, the K /Na ratio was accumulation was higher at 25 and 50 mM NaCl com- negatively affected by salinity at all the concentration. pared to 0 mM NaCl (Fig. 2b). However, no significant The effect was more prominent in shoots, where the differences were observed between 25 and 50 mM NaCl differences between non-saline treatment and either of in control plants. At 25 mM NaCl, only the co-inoculation the salt treatments were highly significant (Fig. 3a). of C. lamellosum with SAB showed a significantly reduced However, microbial treatments did not show significant sodium accumulation in root tissues than all other differences from control at 0 and 25 mM NaCl. At treatments. 50 mM NaCl, only the co-inoculation of C. lamellosum + + The accumulation of chloride ions was significantly in- with SAB showed lower K /Na ratio. In root tissues, at 0 creased in maize shoot tissues with the increase in salinity and 50 mM NaCl, no differences were observed between (Fig. 2c). In shoot tissues, at 0 and 25 mM NaCl, all the the treatments, whereas at 25 mM NaCl, co-inoculation + + treatments showed an increased chloride accumulation treatments showed significantly higher K /Na ratio except with G. margarita. However, at 50 mM NaCl, no (Fig. 3b). significant differences were observed among the treat- ments. In contrast, root tissues exhibited lower chloride Ion transporter gene expression analysis accumulation than control at 0 mM NaCl in all the treat- Ion analysis suggest that microbial colonization affect + + ments (Fig. 2d). At 25 mM NaCl, a single inoculation of tissue K and Na . We therefore tested whether SOS AMF and SAB enhanced accumulation of chloride ions genes expression are regulated by AMF and SAB than control and the co-inoculation treatments showed colonization. AMF colonization significantly altered the + + lower chloride accumulation than control. However, at K and Na accumulation in plants. We have tested the 50 mM NaCl, only co-inoculation of C. lamellosum membrane transporters responsible for K uptake and with SAB showed lower chloride accumulation, other translocation along with Na deposition. Our result treatments exhibited no significant differences as com- showed that the expression of ZmAKT2 gene was pared to control. differentially affected by single and co-inoculation of Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 7 of 13 Table 3 Effect of AMF and SAB co-inoculation on maize root nutrient accumulation under different salinity levels Salt concentration Treatments Total N P K Ca Mg mg / plant d, A e, A b, A c, AB d, AB 0 mM T1 78.8 ± 2.8 13.9 ± 0.9 51.5 ± 5.3 5.6 ± 0.7 4.2 ± 0.3 cd, D cd, D b, D c, E cd, E T2 165.7 ± 26.1 22.1 ± 3.1 53.5 ± 7.4 9.7 ± 0.7 6.4 ± 0.2 bc, G e, G ab, G b, G bc, G T3 250.4 ± 39.6 19.2 ± 3.2d 80.3 ± 17.0 17.0 ± 1.7 8.9 ± 0.8 bcd, J a, J a, J ab, J b, J T4 196.1 ± 18.8 36.7 ± 0.9 106.1 ± 18.6 20.9 ± 2.6 11.2 ± 1.2 ab, M bc, M ab, M ab, M b, MN T5 303.6 ± 27.5 28.6 ± 2.3 77.8 ± 17.9 21.2 ± 2.2 9.9 ± 1.2 a, P ab, P a, P a, P a, P T6 384.7 ± 80.7 30.3 ± 3.0 107.4 ± 17.3 28.1 ± 4.2 16.5 ± 1.4 c, A d, B c, B a, A b, A 25 mM T1 83.6 ± 4.7 7.2 ± 0.5 12.4 ± 3.8 6.9 ± 1.9 5.3 ± 1.8 b, D c, E b, E a, D a, D T2 213.6 ± 42.1 14.4 ± 1.0 34.1 ± 3.7 14.7 ± 1.3 11.5 ± 1.3 b, G bc, GH bc, H a, G ab, G T3 206.9 ± 47.5 16.2 ± 3.3 21.9 ± 4.5 25.0 ± 13.3 8.8 ± 0.8 b, JK bc, K b, K a, K b, K T4 183.9 ± 14.8 20.1 ± 1.8 33.6 ± 8.7 6.7 ± 1.4 5.6 ± 1.0 a, M a, M a, M a, MN a, M T5 341.3 ± 33.3 28.1 ± 2.9 60.1 ± 10.0 18.9 ± 1.3 11.3 ± 1.3 ab, PQ ab, P b, Q a, Q b, Q T6 275.5 ± 20.3 22.4 ± 1.9 32.0 ± 2.9 12.5 ± 1.9 5.2 ± 1.2 b, B c, C b, B c, B b, B 50 mM T1 62.8 ± 3.0 4.6 ± 0.7 5.3 ± 0.9 2.7 ± 0.3 1.4 ± 0.2 a, D ab, E ab, F c, F b, F T2 139.2 ± 21.9 10.5 ± 1.3 13.7 ± 2.1 5.4 ± 1.1 2.4 ± 0.5 a, G bc, H b, H c, G b, H T3 127.3 ± 30.3 8.3 ± 0.8 9.5 ± 2.2 3.5 ± 0.8 1.8 ± 0.5 a, K ab, L ab, K c, K b, K T4 139.5 ± 19.9 10.9 ± 1.5 18.5 ± 4.9 4.5 ± 0.6 3.0 ± 0.5 a, N a, N a, N a, N a, N T5 173.1 ± 22.0 14.0 ± 1.6 24.5 ± 7.0 13.6 ± 1.9 7.2 ± 2.2 a, Q ab, Q ab, Q b, Q a, Q T6 134.8 ± 12.6 11.8 ± 1.4 16.3 ± 7.3 10.0 ± 1.1 3.5 ± 0.6 T1 – control, T2 - Gigaspora margarita S-23, T3 – Claroideoglomus lamellosum S-11, T4 – Pseudomonas koreensis S2CB35, T5 – T2 + T4, T6 – T3 + T4. Plants were subjected to 0 (0.5 dS/m), 25 (2.5 dS/m) or 50 mM NaCl (4.5 dS/m). Different letters indicate significant differences (P < 0.05) among the treatments at each salt level (a, b, c, d, e, f) or among salt levels for each treatment: T1 (A, B, C), T2 (D, E, F), T3 (G, H, I), T4 (J, K, L), T5 (M, N, O) or T6 (P, Q, R). Each value represents the mean of four replicates ± standard error (SE) a c b d + − + + − − Fig. 2 Sodium (Na ) and Chloride (Cl ) content in maize plants. a Na content in shoot, b Na content in root, c Cl content in shoot d Cl content in + − root. See legend for Fig. 1. Each value represents the mean of four replicates (Na ) or three replicates (Cl ) ± standard error (SE) Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 8 of 13 a a + + + + + + Fig. 3 K /Na ratio in maize plants. a K /Na ratio in shoot, b K /Na Fig. 4 Gene expression analysis in maize roots by real-time quantitative ratio in root. See legend for Fig. 1 PCR. a ZmAKT2, b ZmSOS1, c ZmSKOR.See legend forFig. 1 AMF and SAB with increasing salinity (Fig. 4a). At Confocal scanning microscopy 0 mM NaCl, plants inoculated with SAB alone showed The roots of harvested maize plants were observed under significantly lower gene expression compared to control CLSM to confirm the localization of the gfp-tagged SAB and other microbial treatments. At 25 and 50 mM NaCl, strain, P. koreensis. Fluorescent bacterial cells were no significant differences were observed between the observed to be absent in uninoculated control plants treatments. When compared to the salt concentrations, (Fig. 5a). However, plants inoculated with gfp-tagged only plants treated with the co-inoculation of C. lamello- SAB showed that the fluorescent bacterial cells were sum and SAB exhibited increased gene expression (39%) localized on the surface of the roots (Additional file 2: at 25 mM NaCl from 0 mM NaCl; however, the expres- Figure S2). Several SAB were also able to move and sion was reduced significantly at 50 mM NaCl. colonize to inter and intracellular spaces (Fig. 5b and c). The expression of ZmSOS1 and ZmSKOR were nega- SAB P. koreensis S2CB35 efficiently colonized the rhizo- tively affected by salinity (Fig. 4b, c). No significant plane, moved into root tissues, and localized themselves difference in the gene expression of both the genes was to intercellular spaces of root tissues. Furthermore, the observed among different salt concentrations. Each ability of SAB to associate with the spore walls were also treatment exhibited different gene expression at all salt observed (Fig. 5d-i). No SAB colonization was observed concentration for both ZmSOS1 and ZmSKOR genes. on the spore walls of AMF isolated from pots treated with Only plants treated with co-inoculation of C. lamellosum C. lamellosum or G. margarita alone (Fig. 5d, g). Clear and SAB showed significantly higher expression at fluorescent bacterial cells were observed on the spore 25 mM NaCl for ZmSOS1. The higher expression walls of AMF isolated from co-inoculation of AMF and of ZmSKOR was observed in plants treated with SAB treatment pots (Fig. 5e, h,Additional file 3: Figure S3 co-inoculation of C. lamellosum and SAB at 25 mM NaCl and Additional file 4: Figure S4). However, surface ster- compared to 0 mM NaCl. ilized and the broken spores exhibited no endosporic Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 9 of 13 ab c d e f g h i Fig. 5 SAB Pseudomonas koreensis S2CB35-gfp colonization in maize plant roots and association on AMF spore walls. a – control, b – intercellular colonization of SAB, c – Intra cellular colonization of SAB. d, e, f - Claroideoglomus lamellosum S-11, g, h, i - Gigaspora margarita S-23. d and g – Control, e and h – SAB colonization on AMF surface, f and i – No endosporic association. Arrow indicates the gfp-tagged SAB colonization of SAB (Fig. 5f, i) suggesting that the SAB and development [47, 48]. Several studies have reported was limited to the outer surface of AMF spore walls. that salinity reduced growth, leaf area, chlorophyll content, nutrient uptake and photosynthesis [15, 49, 50]. In this Discussion study, dry weight of maize plant decreased with the Plant-microbe symbiosis is an important component for increase in salinity. However, the co-inoculation of plant’s ability to cope with the adverse environmental AMF and SAB significantly increased plant dry weight conditions. Previous studies have demonstrated important under salinity stress. Our results indicate that under mechanisms employed by AMF to promote plant growth salinity, microbial inoculation plays a significant role in under salinity stress [17, 45]. However, these experiments promoting plant growth. were based on the inoculation of AMF alone. In a recent Proline is an important osmoprotectant osmolyte and report by Berta et al. [46], it was demonstrated that the is known to play a vital role in protecting plants from co-inoculation of AMF and soil rhizobia markedly pro- various environmental stresses [51]. Our results demon- moted the growth of maize plant in field conditions than strate that under salinity stress, maize plants accumulated as a single inoculation. Although mycorrhizal colonization a higher amount of proline. However, co-inoculation of is considered nonspecific, it can be enhanced by co-inocu- AMF and SAB significantly reduced proline accumulation lation with mycorrhizal helper bacteria [19, 27]. In the in plants under salinity stress. Previous reports also present work, we analyzed the significance of application of suggested that microbial inoculation decreased the proline two indigenous AMF isolates with a bacterium isolated accumulation in plants [16, 40] under stressful environment. from the surface of AMF spore walls on maize. It has been Mycorrhizal colonization was reported to reduce under reported that salinity negatively affects the plant growth salinity [52]. Similarly, in the present study, mycorrhizal Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 10 of 13 + + colonization was reduced under salt stress; however, the K /Na were found in our study, suggesting that microbial + + co-inoculation of SAB with AMF increased mycorrhizal treatments had a significant impact on K /Na ratio than colonization in all the salt concentration than AMF non-inoculated plant roots under salinity stress. It has + + treatment alone. Our results are in accordance with been suggested that the maintenance of high K /Na Hashem et al. [53], they reported that the co-inoculation ratios in shoots of glycophytes is an important mechanism of AMF with endophytic bacteria increased the mycor- to cope with the salinity stress [60]. In contrast to previous rhizal colonization in Acacia gerrardii under salt stress. reports [9, 29], our result indicates that microbial treatments Although mycorrhizal helper bacteria is known to im- inhibited Cl uptake by plants. Co-inoculation treatments prove fungal growth and colonization efficiency, we found exhibited lower Cl uptake by plant roots under salinity SAB had no positive influence on spore production under stress. A recent study by Elhindi et al. [61]demonstrated salinity stress. that mycorrhizal treated plants showed lower Cl accumula- 2+ 2+ Soil salinity affects the nutrient uptake by plants and tion. Although, a slight increase in Ca and Mg was transport to shoots [54]. Our results indicate that salinity recorded at 25 mM NaCl. The increase in salinity reduced decreased the nutrient uptake by plants. Nitrogen is an the accumulation of these ions. The negative impact of soil 2+ 2+ essential constituent of plant chlorophyll, amino acids, salinity on Ca and Mg uptake was also reported earlier and in the energy transfer compound of ATP (adenosine [47, 52], which is in concordance with our findings. triphosphate). Increased salinity reduced the uptake of Previous reports showed that the inoculation of symbiotic nitrogen; however, inoculation/co-inoculation treatments microbes improves salt tolerance in plants by improving significantly increased the nitrogen uptake by plants, in nutrient uptake [62], antioxidant activity [63], and increased the present study. The phosphate (P) solubilizing microor- synthesis of photosynthetic pigments [53]. Moreover, ion ganisms (PSM) are capable of transforming insoluble P homeostasis is maintained by plants to resist salinity stress. + + into a plant accessible soluble form. AMF is well known It has also been reported that Na /H antiporter overex- for their capability to enhance P uptake by plants. Further, pression affects both salinity tolerance and K nutrition PSM have been reported to increase P uptake by a plant [64]. AKT belongs to the family of plant K inward channel [55]. An increased P uptake by plants was observed in our and is responsible for the uptake of K . AKT2 plays a role study with the use of P solubilizing SAB P. koreensis in sugar loading of the phloem in long distance transport S2CB35; nevertheless, no difference was observed between [65]. On the other hand, the SKOR channel influences the AMF alone treatment and SAB alone treatment. How- xylem loading of K [30]. Our results showed that different ever, a higher P accumulation in plants treated with treatments had different effects on expression of these co-inoculation suggests that the plants might have genes. A highly significant difference was observed at benefited from both AMF and SAB. A similar study by 25 mM NaCl where the plants treated with co-inoculation Battini et al., [27] also reported that co-inoculation of of C. lamellosum and SAB considerably increased the AMF and SAB significantly increased maize plant growth expression of AKT and SKOR. The Na antiporter SOS1 by facilitating the P uptake. has been shown to be involved in the extrusion of Na Furthermore, plants also accumulate inorganic solutes [32]. We found a higher expression of ZmSOS1 gene at such as potassium to maintain osmotic or the turgor 25 mM NaCl in plants co-inoculated with C. lamellosum pressure in addition to organic solute like proline [56] and P. koreensis S2CB35 which correlates with the low + + under salinity stress. A higher level of Na ions present in Na content in the root tissues. Mmycorrhizal treated the soil competes with K ions resulting in an increased plants showed a considerably higher gene expression than + + accumulation of Na ions in plants [57]. K is required for non-inoculated plants and SAB alone. the osmotic balance, has a role in the opening and closing SAB P. koreensis S2CB35 was able to effectively of the stoma, and is an essential factor in protein biosyn- colonize maize root tissues and migrate to inter- and thesis. Giri et al. [58] reported that these functions of K intracellular spaces of root cells. Kost et al. [66] also cannot be substituted with Na ions accumulated in the found that bacteria by utilizing key constituents malate cytosol. In this study, the co-inoculation enhanced the and oxalate of root exudates as sole carbon source were accumulation of K in both root and shoot under salinity able to effectively colonize the root surfaces. The strain stress. According to Estrada et al. [9], root tissues have used in the present study was able to utilize malate as a a higher accumulation of Na than shoots. Cantrell & sole carbon source; however, it was not able to utilize Linderman [59] reported that the accumulated Na in oxalate. The bacterial species, P. koreensis was initially mycorrhizal roots may compartmentalize in cell vacuoles isolated from farming soils in Korea [67]. P. koreensis and in AMF hyphae to prevent translocation to the wasalsoreportedtoexist in variousenvironmental shoots. The co-inoculation treatments at 25 mM NaCl conditions such as in extreme oligotrophic sites [68], and C. lamellosum alone treatment at 50 mM NaCl plant endophytes [69], and heavy metal contaminated showed a lower Na accumulation in roots. High ratios of sites [25]. In the present study, the strain P. koreensis Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 11 of 13 S2CB35 was isolated from the surface of AMF spores. Funding This work was supported by the Strategic Initiative for Microbiomes in CLSM view of AMF spore showed that gfp-tagged SAB Agriculture and Food, Ministry of Agriculture (914004-4), Food and Rural was effectively associated with spore walls of both the Affairs, Republic of Korea. AMF strains. The localization of bacteria on spore have previous been studied [70, 71] and reported to have positive Availability of data and materials All data generated or analyzed during this study are included in this article effect on AMF germination. In addition, diverse bacterial (and its supplementary information files) or are available from the communities were identified to be associated with AMF corresponding author on reasonable request. spores and shown to have multifunctionality [72, 73]. Authors’ contributions GS and TS: conception and design of the work. GS: performed the work. GS and KK: acquisition of data. GS and CS: analyzed the data. GS, CS, SH and TS: Conclusions critical revision of manuscript. GS, CS and TS: wrote the paper. All authors In conclusion, our study indicates that co-inoculation of read and approved the final manuscript. AMF and SAB improved the growth and salt tolerance of maize. Mycorrhizal and bacterial treatments increased Ethics approval and consent to participate + + nutrient uptake by plants and increased ratios of K /Na Not applicable. in root and shoot tissues under salinity stress. A significant Competing interests positive alteration in gene expression of ion homeostasis The authors declare that they have no competing interests. genes was demonstrated by mycorrhizal treatments. Co- inoculation of AMF and SAB exhibited an improved Author details Department of Environmental and Biological Chemistry, College of capability to alleviate inhibitory effects of salinity than Agriculture, Life and Environment Sciences, Chungbuk National University, AMF or SAB alone treatments. SAB was found to be Cheongju, Chungbuk 361-763, Republic of Korea. Horticultural and Herbal associated with the spore walls of AMF and was local- Crop Environment Division, National Institute of Horticultural and Herbal Science, Wanju, South Korea. Department of Agronomy, Benguet State ized in inter- and intra-cellular spaces of maize roots. University, La Trinidad, 2601 Benguet, Philippines. 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Spore associated bacteria regulates maize root K+/Na+ ion homeostasis to promote salinity tolerance during arbuscular mycorrhizal symbiosis

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Abstract

Background: The interaction between arbuscular mycorrhizal fungi (AMF) and AMF spore associated bacteria (SAB) were previously found to improve mycorrhizal symbiotic efficiency under saline stress, however, the information about the molecular basis of this interaction remain unknown. Therefore, the present study aimed to investigate the response of maize plants to co-inoculation of AMF and SAB under salinity stress. Results: The co-inoculation of AMF and SAB significantly improved plant dry weight, nutrient content of shoot and root tissues under 25 or 50 mM NaCl. Importantly, co-inoculation significantly reduced the accumulation of proline + + + in shoots and Na in roots. Co-inoculated maize plants also exhibited high K /Na ratios in roots at 25 mM NaCl concentration. Mycorrhizal colonization significantly positively altered the expression of ZmAKT2, ZmSOS1, and + + ZmSKOR genes, to maintain K and Na ion homeostasis. Confocal laser scanning microscope (CLSM) view showed that SAB were able to move and localize into inter- and intracellular spaces of maize roots and were closely associated with the spore outer hyaline layer. Conclusion: These new findings indicate that co-inoculation of AMF and SAB effectively alleviates the detrimental + + effects of salinity through regulation of SOS pathway gene expression and K /Na homeostasis to improve maize plant growth. Keywords: Arbuscular mycorrhizal fungi, Spore associated bacteria, Plant-microbe interaction, gfp-tagging, Endophytic localization, Salt stress Background Cl ions are taken up by the plant cells causing toxic effects The salinity of soil is one of the most important concerns, such as damage to cell organelles and plasma membrane, which are increasing progressively worldwide. More than disruption of cell organelles, photosynthesis, and protein 800 million hectares (over 6%) of the world’s total land synthesis [4, 5]. As the majority of crop plants are glyco- area are affected by soil salinity (FAO 2005). Increasing phytes, their tolerance to salinity level beyond the threshold salinization of arable lands adversely affects crop estab- level reduces productivity [6]. Maize is the third most lishment, growth, and development contributing to important cereal crop in the world especially in developing huge losses in productivity [1, 2]. The high concentra- countries [7] and is considered as a salt sensitive cereal tion of salt present in the soil causes both hyper-ionic crop [8, 9]. In maize, Na is a major ion and under salt and hyper-osmotic stress and leads to plant death [3]. stress, it causes ion toxicity in plants [10]. Under prolonged salinity stress, the excessive Na and The interaction between plant roots and salt-tolerant microorganisms helps plants alleviate the deleterious effects * Correspondence: tomsa@chungbuk.ac.kr of salinity. Arbuscular mycorrhizal fungi (AMF) can form a Department of Environmental and Biological Chemistry, College of mutualistic association with the roots of more than 80% of Agriculture, Life and Environment Sciences, Chungbuk National University, the terrestrial plants [11]. AMF have been reported to Cheongju, Chungbuk 361-763, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 2 of 13 enhance plant growth under different salinity levels mechanism involved in the alleviation of salinity stress in [5, 12–14] by enhancing the nutrient acquisition in plants. host plants. The alleviation of salt stress by AMF has been Therefore, this study aimed to investigate the effects of reported through increased osmotic balance, increased the co-inoculation of AMF and SAB on the growth of activity of anti-oxidant enzymes, increased photosynthetic maize plant under salinity stress. The study also evalu- activity [15], increased levels of osmoregulant (proline) ated the association of SAB with AMF spore walls and [16] and enhanced water uptake in plants [17]. In addition localization in plant roots; and analyzed the alteration in to the plant, AMF also interacts with many bacterial the expressions of genes involved in ion homeostasis by species in a natural environment. The interactions between AMF and SAB under salinity stress. AMF and bacteria have been shown to improve mutualistic fungus-host interaction [18]and plantgrowth [19]. Some Methods studies have reported the positive effects of co-inoculation Strains detail of AMF and plant growth promoting (PGP) bacteria on Pseudomonas koreensis S2CB35, a SAB, was isolated from plant growth and nutrient uptake under saline stress con- the spore walls of AMF (Gigasporaceae) and demonstrated ditions [20, 21]. Furthermore, many soil microorganisms spore association characteristics as described earlier [35]. and plant endophytic bacteria have been studied and The isolated bacterial strain exhibited multiple plant reported to promote plant growth under various environ- growth-promoting characteristics, such as reduced ethylene mental conditions [22–25]. In our recent study [26], we stress and improved early growth of maize under salt stress found that AMF spore associated bacteria significantly [26]. Two AMF strains, Gigaspora margarita S–23 and reduced ethylene stress level and improved maize seedling Claroideoglomus lamellosum S–11, were used in the growth. Co-inoculation of AMF and mycorrhizosphere present study. They were isolated from a salt affected bacteria increased maize plant growth by enhancing AMF coastal reclamation land of Saemangeum in South hyphal length and facilitating P uptake [27]. However, the Korea and propagated for the mass multiplications by a define mechanisms by which the microbes alleviate salt single spore mass production technique [36]. The detail stress in plants remain unclear. of the strains and protein maker used in this study are Due to the similar physiochemical structure of Na giveninTable 1. + + and K , under salt stress, the excess of Na osmoticum competes for K entry into the symplast, at the transport Green fluorescent protein (gfp)-tagging of the bacterial + + sites. The large cytosolic Na ions compete for K binding strain sites and crucially restricts the metabolic activities that In order to monitor the activity of SAB, the strain was + + require K . The K ion is a key component in the cytosol tagged with GFP before inoculation. The insertion of the as it plays a critical role in protein synthesis, activation of mini-Tn5 gusA::gfp cassette into pB10 was performed by enzymes and photosynthesis, turgor maintenance and introducing Escherichia coli gene Tn5 gusA gfp cassette stomatal movement [28]. AMF is known to selectively (pFAJ1820) [37] into strain P. koreensis S2CB35 by + 2+ uptake K and Ca , which act as osmotic equivalents triparental mating with the helper plasmid pRK2013 of as they avoid the uptake of toxic Na [29]. However, E. coli HB101. The transformants were selected on a the molecular mechanisms of regulation of uptake of K half-strength nutrient agar medium supplemented with + − 1 and exclusion of Na in plants by microbial inoculation kanamycin at 50 μgmL . The presence of GFP in the remain to be elucidated. The salt overly sensitive (SOS) purified transformants was confirmed by PCR amplifica- signaling pathway plays a significant role in maintaining tion using following primers: YL065 (F) 5’ GCGATGTTA + + ion homeostasis by regulating Na and K transport at the ATGGGCAAAAA-3′ and YL066 (R) 5’-TCCATGCCA plasma membrane and tonoplast. The key genes respon- TGTGTAATCCT-3′. The thermal cycling program for sible for ion homeostasis are SOS1, SKOR,and AKT2 [30]. the amplification consisted of initial denaturation at 94 °C Among these, SOS1 is widely studied for its ability to for 3 min, followed by 35 cycles at 94 °C for 30 s, extrude Na and control xylem loading for a long-distance annealing at 56 °C for 1 min, and extension at 72 °C for Na transport [31, 32]. SKOR is involved in the transloca- 1 min, with the final extension at 72 °C for 10 min [38]. tion of K toward shoots through xylem [33]. Further, the The resulting amplicon of 650-bp was confirmed by gel phloem expressing K channel, AKT2, is also involved in electrophoresis. The relative fluorescence activity of the translocation of K in shoots [34]. A previous study gfp-mutant derivatives was analyzed using a flow cytometer conducted by Estrada et al. (2013) reported that these (FACScalibur) equipped with an air-cooled argon-ion laser genes are differentially regulated by AMF regulating ion emitting at 488 nm (15 mW) [39]. Single gfp-derivative homeostasis in plants under salt stress. Moreover, eluci- of P. koreensis S2CB35 was differentiated based on cell dating the gene expression regulated by endophytic morphology, colony appearance, and growth rate char- bacteria might provide broad insights into the molecular acteristics of the wild type. Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 3 of 13 Table 1 Bacterial strains and plasmids used in the present study Plasmid/strain Genotype or other relevant characteristics Reference Escherichia coli S17–1 Vector for plasmid pFAJ1820 Xi et al. [37] Escherichia coli HB101 Helper bacteria containing pRK2013 plasmid Xi et al. [37] P. koreensis S2CB35 PGP-SAB (Genbank number: KM507143) Selvakumar et al. [26] P. koreensis S2CB35-gfp gfp-tagged mutant representative of P. koreensis S2CB35 This study Gigaspora margarita S-23 Propagated AMF (Genbank number: KP677599) Selvakumar et al. [36] Claroideoglomus lamellosum S-11 Propagated AMF (Genbank number: KP677595) Selvakumar et al. [36] pRK2013 Mobilizing plasmid Figurski & Helinski [74] pFAJ1820 pUT mini Tn5gusA-pgfp Xi et al. [37] Soil analysis and seedling preparation which was obtained from 75 g of non-autoclaved AMF The soil sample was collected from low salt affected inoculum, whereas G. margarita inoculated pots received reclamation land of Saemangeum, South Korea. The extracts of C. lamellosum and vice versa. Equally grown physicochemical properties of soil were analyzed using 7-day-old maize seedlings were transplanted into pots the standard laboratory protocols. The pH of soil was 6.0, containing 2.5 kg of soil and maintained for 44 days after with electrical conductivity (EC) value of 0.34 dS/m, organic transplantation (DAT). Each plant was supplemented with matter of 5.5 g/kg, available phosphorus of 40.66 mg/kg, 100 mL of modified Hoagland’s nutrient solution [40] and with content of K, Ca, Mg, and Na measured as 0.56, regularly. 1.0, 2.2, and 0.79 cmolc/kg, respectively. The soil texture For molecular gene expression analysis and confocal was sand 76%, silt 23.2% and clay 0.8%. The maize laser scanning microscopy (CLSM), a separate set was seeds (Zea mays L.) were surface sterilized using 70% prepared with the same treatments. The microbial inocu- ethanol for 1 min, treated for 5 min with 6% NaOCl, lation was applied in the same ratio in 600 mL pots and washed seven times with sterile distilled water. For containing the same soil with an exception that the soil the bacterial treatment, the surface-sterilized seeds were was autoclaved for 3 days consecutively to destroy all imbibed in 10 mL of 0.1 M phosphate buffer (pH 6.8) the microbes present in the soil. Both the experiments were containing 1 × 10 cfu/mL of P. koreensis (S2CB35) for conductedsimultaneouslyunder thesameenvironmental 4 h before the seeds were sown in a seedling tray. For conditions. the control and AMF-alone treatments, the seeds were For seed bacterization, the maize seeds were soaked in treated with 0.1 M phosphate buffer (pH 6.8) with sterile 0.1 M phosphate buffer (pH 6.8) containing 1 × 10 cfu/mL bacteria. of SAB for 2 to 4 h. In addition to seed bacterization, 5 mL of 0.1 M phosphate buffer (pH 6.8) containing Inoculation treatments and salt stress conditions 1×10 cfu/mL of SAB was added to the SAB and In order to study the effects of AMF and SAB on maize co-inoculation treatment pots at 10 and 30 DAT. The growth under NaCl stress, we designed six treatments control and AMF-alone treatments received the same groups designated as T1 for non-treatment control and amount of bacterial culture with the exception that it T2, T3, T4, T5, and T6 for treatments with G. margarita, was autoclaved at 121 °C for 15 min before inoculation. C. lamellosum, SAB, G. margarita + SAB and C. lamello- Salt stress was produced with three different NaCl sum + SAB, respectively, and each one irrigated with three concentrations (0 mM, 25 mM, and 50 mM) at 23 different concentrations of NaCl (0 mM, 25 mM, and DAT. To avoid osmotic shock, NaCl stress was induced 50 mM). The pot experiment was performed in a com- gradually by adding 10 mM and 15 mM to each pot after pletely randomized block design with four replications. every alternate day, and the desired salt concentration was Each pot was filled with 2.5 kg of soil, and the mycorrhizal achieved after 5 days. Leaching of water from the pots was treatment pots received 75 g (3%) of AMF inoculum (each prevented by maintaining the soil water to a level below AMF inoculum containing approximately 200 spores the field capacity at all the times. The maize plants were and 30 root bits), which was added 1 cm below the soil grown for another 15 days under the salt stress condition surface. The control and SAB treatments received 75 g and then harvested. The soil EC value measured at the of autoclaved AMF inoculum to maintain the same nutrient time of harvest for NaCl stress of 0 mM, 25 mM, and content. In addition, to maintain the similar bacterial popu- 50 mM were 0.49 ± 0.09 dS/m, 2.52 ± 0.35 dS/m, and lation in all of the treatments, control, and SAB treatments 4.50 ± 0.12 dS/m, respectively. At the end of the experiment, received 70 mL of soil extract of each AMF inoculum, the plants were harvested carefully, washed in distilled water, Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 4 of 13 separated into leaves, shoots, and roots and were used for GTCCACTTCACTACAC-3′). cDNAs that originated the analysis of different parameters. from three different biological samples were used for each gene analysis. Alpha tubulin (gi:450292) and polyubiquitin Determination of mineral nutrients (gi:248338) were used as the internal controls for the The biomass or dry weight of the shoots was determined normalization of data. All experiments were done in tripli- after oven drying at 70 °C for at least 48 h. The proline cate with three repeats. content of the leaf was estimated according to the method described by Bates et al. [41]. The total nitrogen Preparation of plant root and spore samples for confocal accumulation in the plants was measured using a Kjeldahl microscopy analyzer (K9860 Kjeldahl Analyzer, Hanon Instruments). For confocal scanning laser microscopy (CLSM), the The available phosphorus was determined using the root and soil samples were used from the 600 mL pot vanadate-molybdate method, and the Ca, Mg, Na and K experiment. The fresh root samples removed from SAB concentrations were estimated using inductively coupled treated plant were washed in sterile distilled water and plasma optical emission spectrometry (ICP-OES). dried on a blotting paper. The roots were surface-steril- Chloride anions were determined in an aqueous ized and aseptically sectioned with sterile scalpel blades. extraction obtained from 0.4 g of dry plant material using The sections were mounted on a slide using fluores- 20 mL of deionized water. The extract was shaken for 2 h cence mounting medium under a coverslip. For the and then filtered through a Whatman number 2 filter spores, the isolated spores were either mounted directly paper and a 0.45 μm nylon membrane filter (Millipore). or after surface sterilization with 2% chloramine T and The diluted filtrate was then injected into an ion-exchange 100 μg/mL streptomycin for 30 min. The microscopic ob- chromatography system (Metrohm) packed with anion servation of root and spore samples were performed using separation column Metrosep A Supp 5. a Leica TCS SP2 confocal system (Leica Microsystems Hei- delberg GmbH) equipped with an Ar laser (gfp: Mycorrhizal development excitation, 488 nm; emission filter BP, 500 to 530). Image The root samples were washed with tap water to remove acquisitions were performed under the objectives 20× and the adhering soils, and the roots were cut into the pieces of 40× (N.A. approximately 0.75) and were processed using 1 cm length and stained with 0.05% trypan blue as per the the Zen lite 2012 (blue edition). method described by Phillips & Hayman [42]. The mycor- rhizal root colonization (M%), colonization frequency (F%) Statistical analysis and arbuscules abundances in the whole root system (A%) The data were statistically analyzed using analysis of were calculated according to Trouvelot et al. [43]. The variance (ANOVA) for a completely randomized block isolation of AMF spores from 50 g of soil was carried design with SAS package 9.4 software and the differences out by wet sieving and decanting method [44]. in means were determined by the least significant differ- ences (LSD). Duncan’s multiple-range test was performed Quantitative real time PCR at P ≤ 0.05 on each of the significant variables measured. The maize root samples collected from the 600 mL pot P values less than 0.05 were considered as statistically experiment plants were washed under running water significant. and then rinsed three times with distilled water and frozen in liquid nitrogen, ground and stored at − 80 °C. Total Results RNA was extracted using the RNeasy plant mini kit Plant growth, proline content and mycorrhizal (Qiagen, Valencia, CA, USA) from the root samples stored parameters at − 80 °C. cDNA was synthesized using Superscript III first Microbial inoculation effect on maize plant growth was strand synthesis system (Invitrogen). The gene expression assessed. A significantly negative effect of salinity on analyses were carried out by quantitative reverse transcrip- growth of the plant was observed in all the treatments tion (qRT)-PCR using CFX96 Real-Time System (Bio-Rad and a more prominent effect was evident at the highest Laboratories, München, Germany) with SYBR Green salt concentration of 50 mM NaCl. Treatments with master mix (iQ SYBR Green Supermix, Bio-Rad). Specific AMF and SAB significantly increased the dry weight of primers were designed by Estrada et al. [9] and used to the maize as compared to the control in all salt concen- analyze the genes: ZmAKT2,For (5′-CCTCAAGCATC tration (Fig. 1a). Among the microbial treatments, co- AGGTCGAGA-3′)and ZmAKT2, Rev. (5′-CTCTGTAAT inoculation of C. lamellosum with SAB significantly CTTCCTGGACG-3′), ZmSKOR, For (5′-TCAGATCCA increased plant dry weight at 25 and 50 mM NaCl. Further, AGATGTCCCAG-3′)and ZmSKOR, Rev. (5′-TTCGTA with increasing salt concentration, the corresponding TCCTCTTAACGCAG-3′), ZmSOS1, For (5′-GCTTGTC increase in proline content was observed in all the treat- ACATACTTCACAG-3′)and ZmSOS1,Rev.(5′-ACTT ments (Fig. 1b). However, at 0 mM NaCl, no significant Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 5 of 13 ac bd Fig. 1 AMF and SAB co-inoculation effect on plant growth and mycorrhizal development. a Plant dry weight, b Leaf proline content, c AMF spore count d Mycorrhizal colonization. T1 – control, T2 - Gigaspora margarita S-23, T3 – Claroideoglomus lamellosum S-11, T4 – Pseudomonas koreensis S2CB35, T5 – T2 + T4, T6 – T3 + T4. Plants were subjected to 0 (0.5 dS/m), 25 (2.5 dS/m) or 50 mM NaCl (4.5 dS/m). Different letters indicate significant differences (P < 0.05) among the treatments at each salt level (a, b, c, d, e, f) or among salt levels for each treatment: T1 (A, B, C), T2 (D, E, F), T3 (G, H, I), T4 (J, K, L), T5 (M, N, O) or T6 (P, Q, R). Each value represents the mean of four replicates ± standard error (SE) differences in leaf proline content were observed between uptake in both shoot and root of maize was estimated treatments and control. At 25 mM NaCl, co-inoculation with microbial treatments in all salt levels (Tables 2 with AMF and SAB or C. lamellosum alone treatment and 3). Single and co-inoculation of AMF and SAB significantly reduced the proline content. At 50 mM NaCl, significantly increased total-N and phosphorous in both co-inoculation of C. lamellosum with SAB and only SAB shoot and root at all salt levels. A significantly higher treatments significantly reduced the proline content. potassium uptake was observed for co-inoculation of The effect of salinity on mycorrhizal spore count is shown AMF and SAB in both shoot and root tissues; however, at in Fig. 1c. The increase in salinity reduced AMF spore 50 mM of NaCl, G. margarita with SAB co-inoculation in count. At 0 mM NaCl, the co-inoculation of AMF and shoot and C. lamellosum with SAB co-inoculation in root SAB significantly increased spore count than AMF alone were not significantly different from control. A significantly treatment. However, no significant differences were higher calcium accumulation in shoots was observed for observed between the AMF and AMF co-inoculation with AMF andSAB co-inoculatedplants at0and50mMNaCl. SAB treatments at 25 and 50 mM of NaCl concentration. In root, co-inoculation of AMF and SAB significantly Mycorrhizal colonization in maize roots was negatively improved calcium accumulation at 0 and 50 mM NaCl. affected by increasing salinity (Fig. 1d). A significantly Furthermore, co-inoculation treatment showed signifi- high mycorrhizal colonization was observed with co-in- cantly higher magnesium accumulation in shoots except at oculation of AMF and SAB in all salt concentrations 50 mM NaCl, whereas in roots, co-inoculation treatment compared to AMF alone treatment. Likewise, co-inoculation significantly improved magnesium accumulation at all salt of AMF and SAB exhibited significantly high colonization levels except for C. lamellosum with SAB co-inoculation frequency and arbuscules abundance than AMF alone treatment at 50 mM NaCl. treatment (Additional file 1: Figure S1). Sodium and chloride uptake Nutrient accumulation Under salt stress, plants take up more sodium ion than The efficiency of nutrient uptake by plants under salt potassium. A significant increase in sodium accumula- stress shows the degree of plant response to such stress. tion in maize shoots was observed with the increase in The highest salinity at 50 mM of NaCl concentration salinity (Fig. 2a). At 0 and 25 mM NaCl, no significant significantly lowered the nutrient uptake by plants in all difference was observed between the treatments and the treatments. However, a significantly increased nutrient control. However, at 50 mM NaCl, the co-inoculation of Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 6 of 13 Table 2 Effect of AMF and SAB co-inoculation on maize shoot nutrient accumulation under different salinity levels Salt concentration Treatments Total N P K Ca Mg mg / plant c, A c, A b, A c, A b, A 0 mM T1 819.6 ± 64.3 68.3 ± 6.8 470.4 ± 46.3 40.0 ± 4.9 32.9 ± 1.5 bc, D b, D b, D bc, DE b, DE T2 1368.4 ± 247.1 115.3 ± 11.2 544.5 ± 50.4 45.9 ± 7.0 41.4 ± 5.1 bc, G b, G b, G bc, H b, G T3 1362.9 ± 185.3 113.1 ± 9.9 499.4 ± 74.1 41.1 ± 6.5 42.6 ± 6.1 bc, J b, J ab, J bc, JK b, K T4 1342.7 ± 101.9 133.0 ± 16.9 597.2 ± 137.8 42.0 ± 11.9 36.9 ± 7.1 a, M a, M a, M b, M a, M T5 1997.7 ± 311.2 174.4 ± 15.4 838.0 ± 49.3 69.4 ± 5.7 64.3 ± 3.3 ab, P a, P a, P a, P a, P T6 1921.7 ± 68.9 180.1 ± 9.1 817.0 ± 57.6 96.3 ± 11.2 69.6 ± 2.2 c, AB d, A d, A b, A b, A 25 mM T1 808.3 ± 40.5 66.3 ± 1.1 425.2 ± 11.0 47.0 ± 5.7 46.2 ± 2.3 ab, D bc, D cd, D ab, D ab, D T2 1415.6 ± 96.6 101.8 ± 7.8 470.1 ± 63.5 63.3 ± 8.6 58.7 ± 8.2 b, G c, G bcd, G a, G ab, G T3 1218.6 ± 142.3 98.3 ± 3.2 566.4 ± 28.9 82.1 ± 13.8 66.9 ± 9.4 ab, J b, J abc, J ab, J ab, J T4 1441.1 ± 133.9 129.8 ± 10.5 617.8 ± 44.3 67.3 ± 7.3 66.8 ± 5.8 a, M b, N ab, N ab, M a, M T5 1729.2 ± 117.8 130.6 ± 5.7 697.1 ± 77.9 75.4 ± 5.3 71.0 ± 7.0 a, P a, P a, P a, P a, P T6 1765.1 ± 123.7 161.5 ± 16.5 757.1 ± 70.0 87.4 ± 10.1 78.4 ± 5.7 b, B d, B b, B b, A a, A 50 mM T1 553.4 ± 78.9 50.4 ± 2.2 295.4 ± 31.4 30.5 ± 3.9 42.0 ± 12.5 b, E c, E b, E b, E a, E T2 768.4 ± 24.3 67.8 ± 4.4 287.2 ± 23.3 31.1 ± 2.2 31.9 ± 1.8 b, H bc, H b, H b, H a, G T3 764.1 ± 71.2 72.6 ± 2.9 274.1 ± 18.8 30.2 ± 1.3 50.3 ± 17.8 b, K c, K b, K b, K a, K T4 734.2 ± 98.9 69.7 ± 5.7 304.6 ± 20.7 29.8 ± 4.2 33.2 ± 4.6 a, M b, O b, O a, N a, N T5 1066.9 ± 21.5 87.5 ± 4.1 352.3 ± 12.2 50.6 ± 3.7 46.1 ± 1.6 a, Q a, Q a, Q a, P a, P T6 1265.4 ± 170.4 110.5 ± 11.6 467.4 ± 40.8 63.4 ± 10.8 60.7 ± 8.6 T1 – control, T2 - Gigaspora margarita S-23, T3 – Claroideoglomus lamellosum S-11, T4 – Pseudomonas koreensis S2CB35, T5 – T2 + T4, T6 – T3 + T4. Plants were subjected to 0 (0.5 dS/m), 25 (2.5 dS/m) or 50 mM NaCl (4.5 dS/m). Different letters indicate significant differences (P < 0.05) among the treatments at each salt level (a, b, c, d, e, f) or among salt levels for each treatment: T1 (A, B, C), T2 (D, E, F), T3 (G, H, I), T4 (J, K, L), T5 (M, N, O) or T6 (P, Q, R). Each value represents the mean of four replicates ± standard error (SE) + + C. lamellosum with SAB significantly enhanced sodium K /Na ratios + + accumulation in a shoot. In root tissues, the sodium In both shoots and roots of maize, the K /Na ratio was accumulation was higher at 25 and 50 mM NaCl com- negatively affected by salinity at all the concentration. pared to 0 mM NaCl (Fig. 2b). However, no significant The effect was more prominent in shoots, where the differences were observed between 25 and 50 mM NaCl differences between non-saline treatment and either of in control plants. At 25 mM NaCl, only the co-inoculation the salt treatments were highly significant (Fig. 3a). of C. lamellosum with SAB showed a significantly reduced However, microbial treatments did not show significant sodium accumulation in root tissues than all other differences from control at 0 and 25 mM NaCl. At treatments. 50 mM NaCl, only the co-inoculation of C. lamellosum + + The accumulation of chloride ions was significantly in- with SAB showed lower K /Na ratio. In root tissues, at 0 creased in maize shoot tissues with the increase in salinity and 50 mM NaCl, no differences were observed between (Fig. 2c). In shoot tissues, at 0 and 25 mM NaCl, all the the treatments, whereas at 25 mM NaCl, co-inoculation + + treatments showed an increased chloride accumulation treatments showed significantly higher K /Na ratio except with G. margarita. However, at 50 mM NaCl, no (Fig. 3b). significant differences were observed among the treat- ments. In contrast, root tissues exhibited lower chloride Ion transporter gene expression analysis accumulation than control at 0 mM NaCl in all the treat- Ion analysis suggest that microbial colonization affect + + ments (Fig. 2d). At 25 mM NaCl, a single inoculation of tissue K and Na . We therefore tested whether SOS AMF and SAB enhanced accumulation of chloride ions genes expression are regulated by AMF and SAB than control and the co-inoculation treatments showed colonization. AMF colonization significantly altered the + + lower chloride accumulation than control. However, at K and Na accumulation in plants. We have tested the 50 mM NaCl, only co-inoculation of C. lamellosum membrane transporters responsible for K uptake and with SAB showed lower chloride accumulation, other translocation along with Na deposition. Our result treatments exhibited no significant differences as com- showed that the expression of ZmAKT2 gene was pared to control. differentially affected by single and co-inoculation of Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 7 of 13 Table 3 Effect of AMF and SAB co-inoculation on maize root nutrient accumulation under different salinity levels Salt concentration Treatments Total N P K Ca Mg mg / plant d, A e, A b, A c, AB d, AB 0 mM T1 78.8 ± 2.8 13.9 ± 0.9 51.5 ± 5.3 5.6 ± 0.7 4.2 ± 0.3 cd, D cd, D b, D c, E cd, E T2 165.7 ± 26.1 22.1 ± 3.1 53.5 ± 7.4 9.7 ± 0.7 6.4 ± 0.2 bc, G e, G ab, G b, G bc, G T3 250.4 ± 39.6 19.2 ± 3.2d 80.3 ± 17.0 17.0 ± 1.7 8.9 ± 0.8 bcd, J a, J a, J ab, J b, J T4 196.1 ± 18.8 36.7 ± 0.9 106.1 ± 18.6 20.9 ± 2.6 11.2 ± 1.2 ab, M bc, M ab, M ab, M b, MN T5 303.6 ± 27.5 28.6 ± 2.3 77.8 ± 17.9 21.2 ± 2.2 9.9 ± 1.2 a, P ab, P a, P a, P a, P T6 384.7 ± 80.7 30.3 ± 3.0 107.4 ± 17.3 28.1 ± 4.2 16.5 ± 1.4 c, A d, B c, B a, A b, A 25 mM T1 83.6 ± 4.7 7.2 ± 0.5 12.4 ± 3.8 6.9 ± 1.9 5.3 ± 1.8 b, D c, E b, E a, D a, D T2 213.6 ± 42.1 14.4 ± 1.0 34.1 ± 3.7 14.7 ± 1.3 11.5 ± 1.3 b, G bc, GH bc, H a, G ab, G T3 206.9 ± 47.5 16.2 ± 3.3 21.9 ± 4.5 25.0 ± 13.3 8.8 ± 0.8 b, JK bc, K b, K a, K b, K T4 183.9 ± 14.8 20.1 ± 1.8 33.6 ± 8.7 6.7 ± 1.4 5.6 ± 1.0 a, M a, M a, M a, MN a, M T5 341.3 ± 33.3 28.1 ± 2.9 60.1 ± 10.0 18.9 ± 1.3 11.3 ± 1.3 ab, PQ ab, P b, Q a, Q b, Q T6 275.5 ± 20.3 22.4 ± 1.9 32.0 ± 2.9 12.5 ± 1.9 5.2 ± 1.2 b, B c, C b, B c, B b, B 50 mM T1 62.8 ± 3.0 4.6 ± 0.7 5.3 ± 0.9 2.7 ± 0.3 1.4 ± 0.2 a, D ab, E ab, F c, F b, F T2 139.2 ± 21.9 10.5 ± 1.3 13.7 ± 2.1 5.4 ± 1.1 2.4 ± 0.5 a, G bc, H b, H c, G b, H T3 127.3 ± 30.3 8.3 ± 0.8 9.5 ± 2.2 3.5 ± 0.8 1.8 ± 0.5 a, K ab, L ab, K c, K b, K T4 139.5 ± 19.9 10.9 ± 1.5 18.5 ± 4.9 4.5 ± 0.6 3.0 ± 0.5 a, N a, N a, N a, N a, N T5 173.1 ± 22.0 14.0 ± 1.6 24.5 ± 7.0 13.6 ± 1.9 7.2 ± 2.2 a, Q ab, Q ab, Q b, Q a, Q T6 134.8 ± 12.6 11.8 ± 1.4 16.3 ± 7.3 10.0 ± 1.1 3.5 ± 0.6 T1 – control, T2 - Gigaspora margarita S-23, T3 – Claroideoglomus lamellosum S-11, T4 – Pseudomonas koreensis S2CB35, T5 – T2 + T4, T6 – T3 + T4. Plants were subjected to 0 (0.5 dS/m), 25 (2.5 dS/m) or 50 mM NaCl (4.5 dS/m). Different letters indicate significant differences (P < 0.05) among the treatments at each salt level (a, b, c, d, e, f) or among salt levels for each treatment: T1 (A, B, C), T2 (D, E, F), T3 (G, H, I), T4 (J, K, L), T5 (M, N, O) or T6 (P, Q, R). Each value represents the mean of four replicates ± standard error (SE) a c b d + − + + − − Fig. 2 Sodium (Na ) and Chloride (Cl ) content in maize plants. a Na content in shoot, b Na content in root, c Cl content in shoot d Cl content in + − root. See legend for Fig. 1. Each value represents the mean of four replicates (Na ) or three replicates (Cl ) ± standard error (SE) Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 8 of 13 a a + + + + + + Fig. 3 K /Na ratio in maize plants. a K /Na ratio in shoot, b K /Na Fig. 4 Gene expression analysis in maize roots by real-time quantitative ratio in root. See legend for Fig. 1 PCR. a ZmAKT2, b ZmSOS1, c ZmSKOR.See legend forFig. 1 AMF and SAB with increasing salinity (Fig. 4a). At Confocal scanning microscopy 0 mM NaCl, plants inoculated with SAB alone showed The roots of harvested maize plants were observed under significantly lower gene expression compared to control CLSM to confirm the localization of the gfp-tagged SAB and other microbial treatments. At 25 and 50 mM NaCl, strain, P. koreensis. Fluorescent bacterial cells were no significant differences were observed between the observed to be absent in uninoculated control plants treatments. When compared to the salt concentrations, (Fig. 5a). However, plants inoculated with gfp-tagged only plants treated with the co-inoculation of C. lamello- SAB showed that the fluorescent bacterial cells were sum and SAB exhibited increased gene expression (39%) localized on the surface of the roots (Additional file 2: at 25 mM NaCl from 0 mM NaCl; however, the expres- Figure S2). Several SAB were also able to move and sion was reduced significantly at 50 mM NaCl. colonize to inter and intracellular spaces (Fig. 5b and c). The expression of ZmSOS1 and ZmSKOR were nega- SAB P. koreensis S2CB35 efficiently colonized the rhizo- tively affected by salinity (Fig. 4b, c). No significant plane, moved into root tissues, and localized themselves difference in the gene expression of both the genes was to intercellular spaces of root tissues. Furthermore, the observed among different salt concentrations. Each ability of SAB to associate with the spore walls were also treatment exhibited different gene expression at all salt observed (Fig. 5d-i). No SAB colonization was observed concentration for both ZmSOS1 and ZmSKOR genes. on the spore walls of AMF isolated from pots treated with Only plants treated with co-inoculation of C. lamellosum C. lamellosum or G. margarita alone (Fig. 5d, g). Clear and SAB showed significantly higher expression at fluorescent bacterial cells were observed on the spore 25 mM NaCl for ZmSOS1. The higher expression walls of AMF isolated from co-inoculation of AMF and of ZmSKOR was observed in plants treated with SAB treatment pots (Fig. 5e, h,Additional file 3: Figure S3 co-inoculation of C. lamellosum and SAB at 25 mM NaCl and Additional file 4: Figure S4). However, surface ster- compared to 0 mM NaCl. ilized and the broken spores exhibited no endosporic Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 9 of 13 ab c d e f g h i Fig. 5 SAB Pseudomonas koreensis S2CB35-gfp colonization in maize plant roots and association on AMF spore walls. a – control, b – intercellular colonization of SAB, c – Intra cellular colonization of SAB. d, e, f - Claroideoglomus lamellosum S-11, g, h, i - Gigaspora margarita S-23. d and g – Control, e and h – SAB colonization on AMF surface, f and i – No endosporic association. Arrow indicates the gfp-tagged SAB colonization of SAB (Fig. 5f, i) suggesting that the SAB and development [47, 48]. Several studies have reported was limited to the outer surface of AMF spore walls. that salinity reduced growth, leaf area, chlorophyll content, nutrient uptake and photosynthesis [15, 49, 50]. In this Discussion study, dry weight of maize plant decreased with the Plant-microbe symbiosis is an important component for increase in salinity. However, the co-inoculation of plant’s ability to cope with the adverse environmental AMF and SAB significantly increased plant dry weight conditions. Previous studies have demonstrated important under salinity stress. Our results indicate that under mechanisms employed by AMF to promote plant growth salinity, microbial inoculation plays a significant role in under salinity stress [17, 45]. However, these experiments promoting plant growth. were based on the inoculation of AMF alone. In a recent Proline is an important osmoprotectant osmolyte and report by Berta et al. [46], it was demonstrated that the is known to play a vital role in protecting plants from co-inoculation of AMF and soil rhizobia markedly pro- various environmental stresses [51]. Our results demon- moted the growth of maize plant in field conditions than strate that under salinity stress, maize plants accumulated as a single inoculation. Although mycorrhizal colonization a higher amount of proline. However, co-inoculation of is considered nonspecific, it can be enhanced by co-inocu- AMF and SAB significantly reduced proline accumulation lation with mycorrhizal helper bacteria [19, 27]. In the in plants under salinity stress. Previous reports also present work, we analyzed the significance of application of suggested that microbial inoculation decreased the proline two indigenous AMF isolates with a bacterium isolated accumulation in plants [16, 40] under stressful environment. from the surface of AMF spore walls on maize. It has been Mycorrhizal colonization was reported to reduce under reported that salinity negatively affects the plant growth salinity [52]. Similarly, in the present study, mycorrhizal Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 10 of 13 + + colonization was reduced under salt stress; however, the K /Na were found in our study, suggesting that microbial + + co-inoculation of SAB with AMF increased mycorrhizal treatments had a significant impact on K /Na ratio than colonization in all the salt concentration than AMF non-inoculated plant roots under salinity stress. It has + + treatment alone. Our results are in accordance with been suggested that the maintenance of high K /Na Hashem et al. [53], they reported that the co-inoculation ratios in shoots of glycophytes is an important mechanism of AMF with endophytic bacteria increased the mycor- to cope with the salinity stress [60]. In contrast to previous rhizal colonization in Acacia gerrardii under salt stress. reports [9, 29], our result indicates that microbial treatments Although mycorrhizal helper bacteria is known to im- inhibited Cl uptake by plants. Co-inoculation treatments prove fungal growth and colonization efficiency, we found exhibited lower Cl uptake by plant roots under salinity SAB had no positive influence on spore production under stress. A recent study by Elhindi et al. [61]demonstrated salinity stress. that mycorrhizal treated plants showed lower Cl accumula- 2+ 2+ Soil salinity affects the nutrient uptake by plants and tion. Although, a slight increase in Ca and Mg was transport to shoots [54]. Our results indicate that salinity recorded at 25 mM NaCl. The increase in salinity reduced decreased the nutrient uptake by plants. Nitrogen is an the accumulation of these ions. The negative impact of soil 2+ 2+ essential constituent of plant chlorophyll, amino acids, salinity on Ca and Mg uptake was also reported earlier and in the energy transfer compound of ATP (adenosine [47, 52], which is in concordance with our findings. triphosphate). Increased salinity reduced the uptake of Previous reports showed that the inoculation of symbiotic nitrogen; however, inoculation/co-inoculation treatments microbes improves salt tolerance in plants by improving significantly increased the nitrogen uptake by plants, in nutrient uptake [62], antioxidant activity [63], and increased the present study. The phosphate (P) solubilizing microor- synthesis of photosynthetic pigments [53]. Moreover, ion ganisms (PSM) are capable of transforming insoluble P homeostasis is maintained by plants to resist salinity stress. + + into a plant accessible soluble form. AMF is well known It has also been reported that Na /H antiporter overex- for their capability to enhance P uptake by plants. Further, pression affects both salinity tolerance and K nutrition PSM have been reported to increase P uptake by a plant [64]. AKT belongs to the family of plant K inward channel [55]. An increased P uptake by plants was observed in our and is responsible for the uptake of K . AKT2 plays a role study with the use of P solubilizing SAB P. koreensis in sugar loading of the phloem in long distance transport S2CB35; nevertheless, no difference was observed between [65]. On the other hand, the SKOR channel influences the AMF alone treatment and SAB alone treatment. How- xylem loading of K [30]. Our results showed that different ever, a higher P accumulation in plants treated with treatments had different effects on expression of these co-inoculation suggests that the plants might have genes. A highly significant difference was observed at benefited from both AMF and SAB. A similar study by 25 mM NaCl where the plants treated with co-inoculation Battini et al., [27] also reported that co-inoculation of of C. lamellosum and SAB considerably increased the AMF and SAB significantly increased maize plant growth expression of AKT and SKOR. The Na antiporter SOS1 by facilitating the P uptake. has been shown to be involved in the extrusion of Na Furthermore, plants also accumulate inorganic solutes [32]. We found a higher expression of ZmSOS1 gene at such as potassium to maintain osmotic or the turgor 25 mM NaCl in plants co-inoculated with C. lamellosum pressure in addition to organic solute like proline [56] and P. koreensis S2CB35 which correlates with the low + + under salinity stress. A higher level of Na ions present in Na content in the root tissues. Mmycorrhizal treated the soil competes with K ions resulting in an increased plants showed a considerably higher gene expression than + + accumulation of Na ions in plants [57]. K is required for non-inoculated plants and SAB alone. the osmotic balance, has a role in the opening and closing SAB P. koreensis S2CB35 was able to effectively of the stoma, and is an essential factor in protein biosyn- colonize maize root tissues and migrate to inter- and thesis. Giri et al. [58] reported that these functions of K intracellular spaces of root cells. Kost et al. [66] also cannot be substituted with Na ions accumulated in the found that bacteria by utilizing key constituents malate cytosol. In this study, the co-inoculation enhanced the and oxalate of root exudates as sole carbon source were accumulation of K in both root and shoot under salinity able to effectively colonize the root surfaces. The strain stress. According to Estrada et al. [9], root tissues have used in the present study was able to utilize malate as a a higher accumulation of Na than shoots. Cantrell & sole carbon source; however, it was not able to utilize Linderman [59] reported that the accumulated Na in oxalate. The bacterial species, P. koreensis was initially mycorrhizal roots may compartmentalize in cell vacuoles isolated from farming soils in Korea [67]. P. koreensis and in AMF hyphae to prevent translocation to the wasalsoreportedtoexist in variousenvironmental shoots. The co-inoculation treatments at 25 mM NaCl conditions such as in extreme oligotrophic sites [68], and C. lamellosum alone treatment at 50 mM NaCl plant endophytes [69], and heavy metal contaminated showed a lower Na accumulation in roots. High ratios of sites [25]. In the present study, the strain P. koreensis Selvakumar et al. BMC Plant Biology (2018) 18:109 Page 11 of 13 S2CB35 was isolated from the surface of AMF spores. Funding This work was supported by the Strategic Initiative for Microbiomes in CLSM view of AMF spore showed that gfp-tagged SAB Agriculture and Food, Ministry of Agriculture (914004-4), Food and Rural was effectively associated with spore walls of both the Affairs, Republic of Korea. AMF strains. The localization of bacteria on spore have previous been studied [70, 71] and reported to have positive Availability of data and materials All data generated or analyzed during this study are included in this article effect on AMF germination. In addition, diverse bacterial (and its supplementary information files) or are available from the communities were identified to be associated with AMF corresponding author on reasonable request. spores and shown to have multifunctionality [72, 73]. Authors’ contributions GS and TS: conception and design of the work. GS: performed the work. GS and KK: acquisition of data. GS and CS: analyzed the data. GS, CS, SH and TS: Conclusions critical revision of manuscript. GS, CS and TS: wrote the paper. All authors In conclusion, our study indicates that co-inoculation of read and approved the final manuscript. AMF and SAB improved the growth and salt tolerance of maize. Mycorrhizal and bacterial treatments increased Ethics approval and consent to participate + + nutrient uptake by plants and increased ratios of K /Na Not applicable. in root and shoot tissues under salinity stress. A significant Competing interests positive alteration in gene expression of ion homeostasis The authors declare that they have no competing interests. genes was demonstrated by mycorrhizal treatments. Co- inoculation of AMF and SAB exhibited an improved Author details Department of Environmental and Biological Chemistry, College of capability to alleviate inhibitory effects of salinity than Agriculture, Life and Environment Sciences, Chungbuk National University, AMF or SAB alone treatments. SAB was found to be Cheongju, Chungbuk 361-763, Republic of Korea. Horticultural and Herbal associated with the spore walls of AMF and was local- Crop Environment Division, National Institute of Horticultural and Herbal Science, Wanju, South Korea. Department of Agronomy, Benguet State ized in inter- and intra-cellular spaces of maize roots. University, La Trinidad, 2601 Benguet, Philippines. 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BMC Plant BiologySpringer Journals

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