Assessing the role of endophytic bacteria in the halophyte Arthrocnemum macrostachyum salt tolerance

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Navarro-Torre, S., Barcia-Piedras, J.M., Mateos-Naranjo, E., Redondo-Gómez, S., Camacho, M., Caviedes, M.A., Pajuelo, E. and Rodríguez-Llorente, I.D. (2017), Assessing the role of endophytic bacteria in the halophyte Arthrocnemum macrostachyum salt tolerance. Plant Biol J, 19: 249-256., which has been published in final form at https://doi.org/10.1111/plb.12521 . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self- Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." RESEARCH PAPER 1 2 Assessing the role of endophytic bacteria in the halophyte 3 Arthrocnemum macrostachyum salt tolerance 4 5 S. Navarro-Torre1,†, J. M. Barcia-Piedras2,3,†, E. Mateos-Naranjo2, S. Redondo-6 Gómez2, M. Camacho3, M. A. Caviedes1, E. Pajuelo1 & I. D. Rodríguez-Llorente1* 7 8 1 Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de 9 Sevilla, Sevilla, Spain 10 2 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de 11 Sevilla, Sevilla, Spain 12 3 IFAPA, Centro Las Torres –Tomejil, Sevilla, Spain 13 14 *Correspondence: I. D. Rodríguez-Llorente, Departamento de Microbiología y 15 Parasitología, Facultad de Farmacia, Universidad de Sevilla, C/Profesor García González 16 2, 41012 Sevilla, Spain. E-mail: [email protected] 17 †These authors contributed equal to this work. 18 19 Total words: 6 823 20 Figures included: 3 21 Tables: 2 22 23 24 25 26 27 28 29 30 31 32 33 ABSTRACT 34 35 There is increasing interest in using halophytes for re-vegetation of salt-affected 36 ecosystems, as well as in understanding their mechanisms of salt tolerance. We 37 hypothesised that bacteria from the phyllosphere of these plants might play a key rolei n 38 the high tolerance to excess salinity. Eight endophytic bacteria belonging to Bacillus and 39 closely related genera were isolated from the phyllosphere of the halophyte 40 Arthrocnemum macrostachyum growing in salty agricultural soils. The presence of plant 41 growth-promoting (PGP) properties, enzymatic activities and tolerance to NaCl was 42 determined. Effects of inoculation on seed germination and adult plant growth under 43 experimental NaCl treatments (0, 510 and 1030 mM NaCl) were studied. Inoculation with 44 a consortium including the best performing bacteria considerably improved the kinetics 45 of germination and final germination percentage of A. macrostachyum seeds. At high 46 NaCl concentrations (1030 mM), inoculation of plants mitigated the effects of high 47 salinity on plant growth and physiological performance and, in addition, this consortium 48 appears to increase the potential of A. macrostachyum to accumulate Na+ in shoots, thus 49 improving Na+ phytoextraction capacity. Bacteria isolated from A. macrostachyum 50 phyllosphere seem to play ani mportant role in plant salt tolerance under stressing salt 51 concentrations. The combined use of A. macrostachyum and its microbiome might 52 enhance plant adaptation and Na+ phytoextraction during restoration of salt-degraded 53 soils. 54 55 56 Keywords: Arthrocnemum macrostachyum, endophytes, halophyte, phyllosphere, salt 57 tolerance 58 59 60 61 62 63 64 65 66 67 INTRODUCTION 68 69 Halophytes can be defined as plants that can survive and reproduce in environments 70 where the salt concentration exceeds200 mM NaCl (Flowers & Colmer 2008), or simply 71 plants that grow exclusively on saline soils (Dansereau 1957). The need for re-vegetation 72 and remediation of ecosystems affected by salinity, a growing problem and one of the 73 main factors influencing the reduction in crop yields, has focused research interest on 74 understanding mechanisms of salt tolerance in halophytes(Shabala 2013).Salt tolerance 75 is a complex trait that involves various biochemical and physiological mechanisms that 76 are also species-specific (Flowers & Colmer 2008; Shabala 2013). These mechanisms 77 include increasing the efficiency of external andi nternal Na+ sequestration in trichomes 78 and vacuoles, changes in trichome shape or regulation of ion channels and transporters, 79 tonoplast antiporters or the development of large vacuoles, often with modified lipid 80 composition (reviewed in Shabala 2013; Hasanuzzaman et al. 2014). Furthermore, 81 halophyte scan generate and accumulate compatible osmolytes that contribute to 82 osmoregulation while preventing prolonged Na+ transport to shoots (Rozema et al. 1981) 83 and show great stomatal control that optimises water use efficiency under extreme salinity 84 (Redondo-Gómez et al. 2010; Shabala 2013). Despite extensive knowledge on the 85 mechanisms of tolerance in halophytes, plant tolerance must also be connected with 86 complex ecological processes, which are highly influenced by microorganisms that 87 colonise the rhizosphere and phyllosphere, the socalled plant microbiome. Although the 88 halophyte microbiome may contribute to plant adaptation and salt tolerance, studies with 89 bacteria inhabiting halophytes are still scarce, and most are merely descriptive, i.e. tried 90 to classify and count microbial species (Ruppel et al. 2013). Bacteria collectively known 91 as plant growth-promoting bacteria (PGPB) have a beneficial effect on plant growth, 92 health and tolerance to different environmental stresses, including contamination with 93 organic compounds, heavy metals or salinity (de Bashan et al. 2012; Glick 2012;Mateos-94 Naranjo et al. 2015; Mesa et al. 2015a,b; Ullah et al.2015). General mechanisms 95 underlying these beneficial effects include, among others, phosphorus solubilisation, 96 nitrogen fixation, iron sequestration, phytohormones (mainly auxins),ACC (1-97 aminocyclopropane-1-carboxylic acid) deaminase synthesis and biofilm formation (Ullah 98 et al. 2015). In this context, several authors reported the use of PGPB to enhance salt 99 tolerance and plant productivity under saline conditions (de Bashan et al. 2012; Ruppel 100 et al. 2013). Nevertheless, there are still few studies describing PGPB application in 101 halophytes to extract solid conclusions and, in addition, these are restricted to rhizosphere 102 bacteria in most cases (Ruppel et al. 2013; Jesús et al. 2015). Therefore, this study was 103 designed and conducted inorder to contribute to filling this gap in knowledge. 104 Arthrocnemum macrostachyum (Moric) C. Koch is a suitable model plant to conduct a 105 thorough study of the role of endophytic bacteria on plant performance under salinity 106 stress, since it is a halophyte that is able to adapt physiologically to a wide range of 107 salinities and grow under extreme hypersaline conditions, even with 1030 mM NaCl, and 108 has a high capacity or accumulation of Na+ (Redondo-Gómez et al. 2010), which makes 109 it a suitable candidate for remediation of salt-affected soils. In the present study we 110 hypothesised that bacteria from the phyllosphere of A. macrostachyum might play a key 111 role in the high salinity tolerance of this species. Thus the specific aims of this study with 112 A. macrostachyum were to: (i) isolate, characterize and select bacterial endophytes with 113 plant growth-promoting(PGP) properties from aerial parts; (ii) investigate how these 114 bacteria affect seed germination; (iii) explore their contribution to growth of adult plants 115 in experimental NaCl treatments ranging from 0 to 1030 mM NaCl and ascertain the 116 extent to which the photosynthetic apparatus determines plant performance; and (iv) 117 determine bacterial influence on Na+ accumulation patterns. 118 119 MATERIAL AND METHODS 120 121 Isolation of endophytic bacteria from A. macrostachyum aerial parts 122 123 Plants of A. macrostachyum were collected from an agricultural soil in Lebrija (Seville, 124 Spain; 36° 540 N, 6° 120 E) in December 2013. Samples of aerial parts from three plants 125 (each sample containing three fragments of plant material of 2–3 cm length) were surface 126 disinfected in 50 ml sterile Falcon tubes in aseptic conditions. First, they were washed 127 with 70% (v/v) ethanol for1 min by shaking and then submerged in sodium 128 hypochlorite1:4 in sterile distilled water for 15 min and gently shaken. Finally, it was 129 washed four times in sterile distilled water. Disinfected samples were crushed separately 130 with a sterile mortar in 1 ml 0.9% sterile saline solution. Aliquots of 100 µl of the resulting 131 mixtures were plated onto five plates containing tryptic soy agar (TSA) medium 132 (Thompson et al. 1993) and five containing TSA medium supplemented with 0.3 M 133 NaCl. Petri dishes were incubated at 28 °C for 72 h and the different colonies based on 134 morphology were isolated. TSA of 0.3 M NaCl was prepared by adding SW30 solution 135 to the distilled water (Mesa et al. 2015a). 136 137 Isolation of DNA and identification of endophytic bacteria by16S rRNA amplification 138 139 Genomic DNA was extracted using the i-genomic BYF DNA Extraction kit (Intron 140 Biotechnology, Korea), according to the manufacturing’ instructions. 16S rRNA 141 amplification was performed using the 16F27 primer (50-142 AGAGTTTGATCMTGGCTCAG-30) and 16R1488 primer (50-143 CGGTTACCTTGTTAGGACTTCACC-30) with the following PCR conditions: initial 144 denaturation at 95 °C for 2 min, 30 cycles of denaturation at 95 °C for 45 s, annealing at 145 58 °C for 45 s, extension at72 °C for 3 min and a final extension at 72 °C for 5 min. 146 16SrRNA sequences were compared with those in the databases using the EzTaxon server 147 (Chun et al. 2007). 16S rRNA sequences were deposited in GenBank under the accession 148 numbers shown in Table 1. 149 150 Amplification of genes related to synthesis of osmoprotectants 151 152 The genes ectBC and betB were amplified. For ectBC gene amplification, Tra3 and CR 153 primers (Reshetnikov et al. 2011) were used, following the PCR conditions described by 154 the authors. This amplification gives PCR products of around 900 bp (Reshetnikov et al. 155 2011). For amplification of betB, degenerate primers betBF (50-156 ATYGGYGCMTGGAACTAYCC-30) and betBR (50-157 ACCGGBCCRAARATYTCYTC-30) were designed based on the sequences of several 158 betB genes from different genera deposited in the NCBI Genebank database (sequences 159 with accession nos. CP000949.1, CP000749.1, CP002447.1, AJ238780.1, JN426961.1, 160 AE014075.1, CP001830.1,EQ973121.1) using CLUSTAL Omega program 161 (http://www.ebi.ac.uk/Tools/msa/clustalo/; Figure S1). PCR conditions were initial 162 denaturation at 93 °C for 2 min, 34 cycles of denaturation at 93 °C for 45 s, annealing at 163 55.7 °C for 1 min, extension at 72 °C for 2 min and a final extension at 72! C for5 min. 164 The PCR product for this amplification was 700 bp, approximately. 165 166 Enzymatic activities and resistance to NaCl 167 168 Pectinase and cellulase activities were examined according to the method described in 169 Elbeltagy et al. (2000). Amylase activity was assessed in starch agar plates incubated for 170 7 days at28 °C and revealed with 10 ml Lugol. Lipase and protease activities were 171 observed as the presence of halos around bacteria after incubation in Tween and casein 172 agars, respectively, for7 days at 28 °C (Prescott 2002). Concerning DNAse activity, 173 bacteria were incubated 7 days at 28 °C in DNA agar plates. Plates were then treated with 174 1 M HCl; halos in dark background were observed in bacteria with DNAse activity. 175 Chitinase activity was assessed as described in Mesa et al. (2015b). Plates were 176 supplemented with 0.3 M NaCl. To determinate resistance to NaCl, bacteria were 177 incubated in TSA plates supplemented with increasing concentrations of salt (adding 178 SW30 solution) at 28 °C for 72 h. 179 180 Plant growth-promoting properties 181 182 All the plant growth-promoting traits of isolated bacterial strains were evaluated in the 183 presence of 0.3 M NaCl. N fixation, bacteria were plated in NFb (nitrogen fixation 184 bromothymol blue), a nitrogen-free medium prepared as described in Dobereiner (1995), 185 and incubated for 72 h at28 °C. Siderophore production was assessed in CAS 186 (chromeazurol S) agar plates (Schwyn & Neilands 1987). Plates werei ncubated for 7 187 days at 28 °C in darkness. Bacteria that produced siderophores had an orange halo. 188 Phosphate solubilization was determinate as the presence of a halo around the bacteria in 189 NBRIP plates (Nautiyal 1999) after 7 days at 28 °C.IAA (indoleacetic acid) production 190 was evaluated in nutrient broth media (Biolife, Italy) supplemented with L-tryptophan 191 (100 mg·l-1) and incubated at 28 °C for 72 h with shaking. Salkowsky reagent (Gordon & 192 Weber 1951) was then added and optical density at 535 nm measured using a 193 spectrophotometer (Lambda25; PerkinElmer, USA). 1-aminocyclopropane-1-car- 194 boxylate (ACC) deaminase activity was assessed as describe in Penrose & Glick (2003). 195 Briefly, isolated strains were incubated in DF salts minimal medium with (NH4)2SO2 as 196 a source of N for 24 h at 28 °C with shaking. An aliquot was then transferred to DF salts 197 minimum medium with 3 mM ACC and incubated for a further 24 h at the same 198 temperature. Finally, biofilm formation was assayed following the method described in 199 del Castillo et al. (2012), starting from a bacterial culture in TSB supplemented with 0.3 200 M NaCl. 201 202 Inoculation of A. macrostachyum seeds and plant growth conditions 203 204 Preparation of bacterial inoculum 205 206 The three strains that showed the best plant beneficial traits were selected for plant 207 inoculation. They were incubated separately in TSB medium supplemented with 0.3 M 208 NaCl for18–24 h at 28 °C with continuous shaking to reach an optical density at 600 nm 209 around 0.9–1.0, corresponding to 108cells·ml-1. Cultures were then centrifuged in 50-ml 210 Falcon tubes at 8000 rpm for 10 min. Pellets were washed with 0.9% saline solution, 211 centrifuged for 10 min in the same conditions and then re-suspended in 5 ml 0.9% saline 212 solution. Finally, cultures were mixed in a 50-ml tube. 213 214 Seed germination experiment 215 216 Seeds of A. macrostachyum were collected in March 2015 in Lebrija (Seville, Spain; 36° 217 540 N, 6° 120 E) from 20 different plants chosen randomly from an anthropic marsh with 218 no tidal influence (mean sea level +2.7 m relative to SHZ) and stored in the dark for 2 219 months at 4 °C until the start of the experiments. In May 2015 a germination experiment 220 was performed to assess the effect of bacterial inoculation on seed germination capacity 221 of A. macrostachyum in presence of 0.9% NaCl. Thus, prior to bacterial inoculation, seeds 222 were surface disinfected with 10% sodium hypochlorite for 10 min and then six times 223 washed with sterile distilled water. Seeds were then divided in two blocks, one was 224 inoculated with the selected bacterial consortium by submerging seeds in 15 ml culture 225 (containing 5 ml of each strain EA1, EA3 and EA8, prepared as described above) for 1 h 226 with shaking, while the second block was not inoculated(submerged in the same culture 227 media without bacteria). Five 25-seed replicates were then placed on 9% agar plates 228 containing 0.9% NaCl (n = 250, five plates with 25 seeds each inoculation treatments) 229 and placed in a growth chamber (AGP-700-HR ESP; Radiber, Spain) with a regime of 10 230 h light (20 °C, 50% RH and 35 µmol·m-2·s-1, 400–700 nm) and 14 h dark (5 °C and 50% 231 RH). Seeds were inspected daily for 20 days and seeds were considered to have 232 germinated after radicle appearance (Redondo-Gómez et al. 2004). Germination kinetics 233 and the final germination percentage were determined. 234 235 Plant growth experiment 236 237 Another set of inoculated and non-inoculated seeds (see above) were germinated on 9% 238 agar plates with regime of 10 h light (20 °C, 50% RH and 35 µmol·m-2·s-1, 400–700 nm) 239 and 14 h dark (5 °C and 50% RH). Seedlings were then transferred to pots with perlite 240 and separated into different trays depending of the inoculation treatment. Pots were 241 maintained at controlled temperature between 21 and 25 °C, 40–60% RH, natural day 242 light of 250 µmol·m-2·s-1 as minimum and 1000 µmol·m-2·s-1 as maximum light and 243 supplemented with 20% Hoagland solution (Hoagland & Arnon 1938) without NaCl 244 supplementation. In September 2015, pots with 4-month-old plants were placed in 245 individual trays and randomly assigned to three salinity treatments (0, 510, 1030 mM 246 NaCl), maintaining the inoculation treatment under which they had grown, i.e. with 247 orwithout bacterial inoculation (n = 54, nine plants 9 three NaCl concentrations 248 inoculation treatments). Thus each plant derived from inoculated seeds was re-inoculated 249 with 200 µl of a culture with the bacterial consortium (prepared as described above), 250 every week for the first 3 weeks and once a month during the experimental period. Saline 251 solutions were established by combining 20% Hoagland solution with the appropriate 252 amount of NaCl. Thus, at the beginning, 3 l of each solution (0, 510, 1030 mM NaCl) 253 were placed in each tray to a depth of1 cm, and during the experiment the levels of the 254 trays were monitored and topped up to the marked level with 20% Hoagland solution 255 (without additional NaCl) to limit changes in NaCl concentration caused by water 256 evaporation from the nutrient solution. In addition, the entire solution (including the 257 NaCl) was changed every week. At the end of the experiment, after 3 months growing 258 with different NaCl concentrations (7-month-old plants), in order to assess the effect of 259 bacterial inoculation on A. macrostachyum response to salinity in terms of growth, 260 photosynthetic response and Na+ accumulation capacity, the following measurements 261 were performed. For growth analysis, at the beginning and the end of the experiment, 262 three and six plants per treatment were harvested and dried at 60 °C for 48 h to estimate 263 total shoot and root dry weights and calculate the relative growth rate (RGR; Redondo-264 Gómez et al. 2010). For photosynthetic response assessment, instantaneous gas exchange 265 and fluorescence measurements were taken on random primary branches of each plant (n 266 = 6) using an in fraredgas analyser in an open system (LI-6400-XT; Li-Cor, Lincoln, NE, 267 USA) and a modulated fluorimeter (FMS-2; Hansatech Instruments, UK), respectively. 268 Thus net photosynthesis rate (AN), stomatal conductance (gs) and intercellular 269 CO2concentration (Ci) were obtained at a light intensity of 1000 µmol·m-2·s-1, 270 REFERENCES 474 475 Abou-Shanab, R.A.I., van Berkum, P., Angle, J.S. (2007). 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Values 712 represent mean ± SE, n = 6. Different letters indicate means that are significantly different 713 from each other (P < 0.05). 714 715 Figure 3. Total sodium (Na) concentration for shoot (A) and root (B) of A. 716 macrostachyum in response to a range of NaCl concentrations and absence (non-717 inoculated) or presence (inoculated) of bacteria for 3 months. Values represent mean ± 718 SE, n = 6. Different letters indicate means that are significantly different from each 719 other (P < 0.05). 720 721 722