ELUCIDATION OF THE PLANT GROWTH –PROMOTING EFFECT OF Hartmannibacter diazotrophicus ON TOLERANCE OF BARLEY TO SALT STRESS Cumulative Dissertation Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of natural sciences (Doctor rerum naturalium - Dr. rer. nat.) Submitted by: M.Sc. JULIAN ALONSO ROJAS BARRETO Justus Liebig University Giessen Research Centre for Biosystems, Land Use and Nutrition (IFZ) Institute of Applied Microbiology Gießen, 2021 The present work was carried out at the Institute for Applied Microbiology, Faculty of Agricultural Sciences, Nutritional Sciences and Environmental Management, Justus-Liebig-University, Gießen during the period from January 2017 to January 2021 under the guidance of Prof. Dr. Sylvia Schnell. 1. Supervisor: Prof. Dr. Sylvia Schnell Institute for Applied Microbiology Justus Liebig University Heinrich-Buff-Ring 26-32, 35392 Gießen, Germany 2. Supervisor: Prof. Dr. Hans-Werner Koyro Institute for Plant Ecology Justus Liebig University Heinrich-Buff-Ring 26, 35392 Gießen, Germany II | S e i t e Declaration I declare that the dissertation here submitted is entirely my own work, written without any illegitimate help by any third party and solely with materials as indicated in the dissertation. I have indicated in the text where I have used texts from already published sources, either word for word or in substance, and where I have made statements based on oral information given to me. At all times during the investigations carried out by me and described in the dissertation, I have followed the principles of good scientific practice as defined in the “Statutes of the Justus Liebig University Gießen for the Safeguarding of Good Scientific Practice”. Date Julian Rojas III | S e i t e TABLE OF CONTENTS List of Abbreviations ............................................................................... 3 Summary ................................................................................................. 6 Zusammenfassung .................................................................................. 7 CHAPTER 1. Introduction and Background .................................................................. 9 1. Introduction and background ....................................................... 10 1.1 Salinity ................................................................................... 10 1.2 Effects of soil salinity on plant growth .......................................... 11 1.3 Plant growth-promoting rhizobacteria (PGPR) ............................... 12 1.3.1 Alleviation of salt stress by PGPR ............................................. 12 1.4 Methylotrophs ......................................................................... 15 1.4.1 Aerobic oxidation of methane to formaldehyde ........................... 16 1.4.2 Methanol dehydrogenase (MDH) .............................................. 19 1.4.3 Expression Regulation of xoxF ................................................. 22 1.5 Hartmannibacter diazotrophicus E19T .......................................... 23 1.6 Barley .................................................................................... 25 1.7 Taxonomical description of a new Spirosoma species ..................... 26 1.8 Comparative genomics .............................................................. 27 1.9 Aim of the study ...................................................................... 27 1.10 References .............................................................................. 28 CHAPTER 2. Effect of Hartmannibacter diazotrophicus and lanthanum on the plant growth and microbial communities of barley grown under salt stress ....................... 44 CHAPTER 3. Spirosoma endbachense sp. nov., isolated from a natural salt meadow .......... 75 CHAPTER 4. Draft Genome Sequences of Spirosoma agri KCTC 52727 and Spirosoma terrae KCTC 52035 ............................................................................................ 90 CHAPTER 5. Screening of bacterial strains with methylotrophic activity lanthanum dependent ............................................................................................... 93 CHAPTER 6. GENERAL DISCUSSION ........................................................................... 106 ACKNOWLEDGMENTS.............................................................................. 115 1 | S e i t e List of peer-reviewed publications Rojas, J., Ambika Manirajan, B., Ratering, S., Suarez, C., Geissler-Plaum, R., & Schnell, S. (2021). Spirosoma endbachense sp. nov., isolated from a natural salt meadow. International Journal of Systematic and Evolutionary Microbiology, 71(1), 1–7. https://doi.org/10.1099/ijsem.0.004601 Rojas, J., Ambika Manirajan, B., Ratering, S., Suarez, C., & Schnell, S. (2020). Draft Genome Sequences of Spirosoma agri KCTC 52727 and Spirosoma terrae KCTC 52035. Microbiology Resource Announcements, 9(23). https://doi.org/10.1128/MRA.00317-20 Rojas, J., Suarez, C., Ratering, S., Krutych, M., Hormaza, A., & Schnell, S. Effect of Hartmannibacter diazotrophicus inoculation and lanthanum on barley growth under saline conditions. Submitted to: Biology and Fertility of Soils JARB designed the experiments and performed data collection, data evaluation and writing of manuscripts drafts. List of conference contributions Poster Rojas, J., Suarez, C., Ratering, S., & Schnell, S. (2018) Plant growth-promoting effect of Hartmannibacter diazotrophicus and lanthanum on barley under saline conditions. 17th International Symposium on Microbial Ecology (ISME17), Session: Plant microbe interactions, 12-17 August 2018, Leipzig, Germany 2 | S e i t e List of Abbreviations 16S rRNA 16S ribosomal RNA ABA Abscisic acid ACC 1-aminociclopropane ACC-deaminase Enzyme 1-aminociclopropane-1-carboxylase aRuMP Assimilatory ribulose monophosphate pathway Asp Aspartic acid ASV Amplicon sequence variations BS Bifidobacterium shunt C1 One-carbon metabolism CDS Coding sequence DDH DNA-DNA hybridization DAP Dihydroxyacetone phosphate DPG 2,3-diphosphoglycerate E19T Hartmanibacter diazotrophicus EDD Entner-Doudoroff pathway EMP Embden-Meyerhof-Parnas pathway E4P Erythrose-4-phosphate FAO Food and Agriculture Organization Fdh Formate dehydrogenase FDH Formate dehydrogenase F6P Fructose 6-phosphate F1,6P Fructose 1,6-bisphosphate G3P Glyceraldehyde 3-phosphate G6P Glucose 6-phosphate 3 | S e i t e He6P 3-hexulose 6-phosphate HKT High-affinity potassium transporter HPi 3-hexulose 6-phosphate isomerase HPR Hydroxypyruvate reductase HPS 3-hexulose 6-phosphate synthase H4FP Folate-linked C1 transfer H4MTP Methanopterin-linked C1 transfer IAA Indole acetic acid KDPG 2-keto-3-deoxy 6-phosphogluconate MCL Malyl coenzyme A lyase MD Malate dehydrogenase MDH Methanol dehydrogenase MMO Methane monooxygenase MtdA Methylene tetrahydromethanopterin dehydrogenase MTK Malate thiokinase mxaF Methanol dehydrogenase-gene NDH2 NADH:quinone oxidoreductase 6PG 6-phosphogluconate PEP phosphoenolpyruvate PGA 3-phosphoglycerate PGPR Plant Growth-Promoting Rhizobacteria Pi Inorganic phosphate PPi Diphosphate PQQ Pyrrolo-quinoline quinone pMMO Particulate form methane monooxygenase REEs Rare earth elements 4 | S e i t e ROS Reactive oxygen species RuMP Ribulose monophosphate pathway Ru5P Ribulose 5-phosphate R5P Ribose 5-phosphate SC Serine cycle S7P Sedoheptulose 7-phosphate sMMO Soluble form Methane monooxygenase STHM Serine hydroxymethyl transferase VOC Volatile organic compounds xoxF Methanol dehydrogenase-gene X5P Xylulose 5-phosphate 5 | S e i t e Summary A novel type of methanol dehydrogenase was recently identified, this dehydrogenase is encoded by the gene xoxF and for its activity requires lanthanum (La3+) as a cofactor. The xoxF-gene was detected in the bacterium Hartmannibacter diazotrophicus which stands out by its activity as a plant growth promoter under saline stress by mechanisms that include production of ACC- deaminase, nitrogen fixation, phosphorus solubilization, among other. Methanol dehydrogenase xoxF activity of H. diazotrophicus was evaluated by its growth on methanol in a mineral medium supplemented with lanthanum and the suitable concentration of lanthanum of optimal growth was determined. Moreover, a greenhouse experiment with barley plants growing in saline soil (NaCl 3%) was reassessed and six experimental treatments were set up namely: Two treatments of barley seeds inoculated with H. diazotrophicus with/without lanthanum amendment, two treatments of seeds with addition of H. diazotrophicus death biomass with/without lanthanum amendment, one treatment of seeds with lanthanum amendment and the controls without amendments respectively. Variations in shoots and roots dry weights, changes in microbial diversity and colonization of H. diazotrophicus in roots and rhizosphere were evaluated. Inoculated plants with/without lanthanum showed significant increases of leave and root dry weights in comparison to controls. Plants growing with only La3+ also exhibited higher leave and root dry weights in contrast to treatments with death biomass addition and the controls. Plants inoculated with dead biomass of H. diazotrophicus with and without lanthanum had no significant effect in plant growth under salt stress compared to non-inoculated plants. Changes in alpha and beta bacterial diversity in communities of rhizosphere and roots were evidenced among the treatments; alpha diversity indicators showed an increase in the number of ASVs (Amplicon sequence variations) for treatments with H. diazotrophicus, whereas beta diversity analysis revealed variation in microbial communities throughout the treatments. Furthermore, for H. diazotrophicus a high colonization capacity of roots and rhizosphere was demonstrated in presence/absence of lanthanum. Furthermore, samples from natural saline environments of soil and waters were collected for bacterial enrichments in liquid mineral medium with methanol and lanthanum in order to isolate potential halotolerant bacteria with methylotrophic 6 | S e i t e activity. Thirty-one bacteria were isolated and among them, one new species of Spirosoma sp. genus was identified and after polyphasic approach was proposed and accepted as Spirosoma endbachense I-24T. The draft genome of S. endbachense I-24T confirmed the position as new species and 4 clusters involved in biosynthesis of ladderane, terpene, polyketide synthese type I and III and non- ribosomal peptide synthese were identified. For Spirosoma agri KCTC 52727T and Spirosoma terrae KCTC 52035T which are the next relatives to S. endbachense I- 24T, draft genome sequences were also assembled. The annotation revealed carbohydrate-active enzymes and secondary metabolite biosynthesis gene clusters as well as alkaline phosphatase, cellulose and amylase activity. The genomes contribute to the genomic knowledge of the members of the genus Spirosoma. Zusammenfassung Vor kurzem wurde ein neuartiger Typ einer Methanol-Dehydrogenase identifiziert. Diese Dehydrogenase wird durch das Gen xoxF kodiert und benötigt für ihre Aktivität Lanthan (La3+) als Cofaktor. Das xoxF-Gen wurde im Bakterium Hartmannibacter diazotrophicus nachgewiesen, das sich durch seine Aktivität als Pflanzenwachstumsförderer unter Salzstress auszeichnet, und zwar durch Mechanismen, die u.a. die Aktivitäten von ACC-Deaminase, Stickstofffixierung und Phosphorsolubilisierung umfassen. Die Methanol-Dehydrogenase xoxF-Aktivität von H. diazotrophicus wurde durch sein Wachstum auf Methanol in einem mit Lanthan supplementierten Mineralmedium untersucht und die geeignete Lanthankonzentration für optimales Wachstum wurde bestimmt. Außerdem wurde ein Gewächshausexperiment mit Gerstenpflanzen, die in salzhaltigem Boden (NaCl 3%) wachsen, durchgeführt und dafür sechs Versuchsgruppen eingerichtet, nämlich: Zwei Versuchsgruppen von Gerstensamen mit H. diazotrophicus beimpften mit/ohne Lanthan-Zusatz, zwei Versuchsansätze von Samen mit Zusatz von abgetöteter Biomasse von H. diazotrophicus mit/ohne Lanthan-Zusatz, ein Versuchsansatz von Samen mit Lanthan-Zusatz und Kontrollen ohne Zusatz. Die Trockengewichte von Blättern und Wurzeln, die Veränderungen der mikrobiellen Diversität und die Kolonisierung von H. diazotrophicus an Wurzeln und in der Rhizosphäre wurden ausgewertet. Inokulierte Pflanzen mit/ohne Lanthan zeigten einen signifikanten Anstieg des Blatt- und Wurzeltrockengewichts im Vergleich zu 7 | S e i t e den Kontrollen. Pflanzen, die nur mit La3+ wuchsen, zeigten ebenfalls ein höheres Blatt- und Wurzeltrockengewicht im Vergleich zu den Versuchsansätzen mit Zusatz von toter Biomasse und den Kontrollen. Pflanzen, die mit toter Biomasse von H. diazotrophicus mit oder ohne Lanthan inokuliert wurden, zeigten keine signifikante Wirkung auf das Pflanzenwachtum unter Salzstress im Vergleich zu nicht- beimpften Pflanzen. Veränderungen der Alpha- und Beta-Diversität der bakteriellen Rhizosphäre- und Wurzelgemeinschaften wurden analysiert; Alpha- Diversitätsindikatoren zeigten eine Zunahme der ASVs (Amplicon sequence variations) für die Versuchsgruppen mit H. diazotrophicus, während die Analyse der Beta-Diversität Unterschiede der mikrobiellen Gemeinschaften entsprechend der Versuchsansätzen zeigte. Für H. diazotrophicus wurde eine hohe Kolonisierungskapazität der Wurzeln und in der Rhizosphäre in Anwesenheit/Abwesenheit von Lanthan gezeigt. Um potenziell halotolerante Bakterien mit methylotropher Aktivität neu zu isolieren wurden Proben aus natürlichen salzhaltigen Umgebungen von Böden und Gewässern gesammelt und für Bakterienanreicherungen in flüssigem Mineralmedium mit Methanol und Lanthan verwendet. Einunddreißig Bakterienstämme wurden isoliert und darunter wurde eine neue Art der Gattung Spirosoma identifiziert und nach einem polyphasischen Ansatz als Spirosoma endbachense I-24T vorgeschlagen und akzeptiert. Das (draft) Genom von S. endbachense I-24T bestätigte die Position der neuen Art und 4 Gengruppen für die Biosynthese von Ladderan, Terpen, Polyketid Synthese Typ I und III sowie eine nicht-ribosomale Peptidsynthetase wurden identifiziert. Für Spirosoma agri KCTC 52727T and Spirosoma terrae KCTC 52035T als nächst verwandte Arten von Spirosoma endbachense wurden ebenfalls die Genome sequenziert und assembiert (draft genomes). Die Annotation ergab Gene für kohlenhydrataktive Enzyme und Gencluster für die Biosynthese von Sekundärmetaboliten sowie für alkaline Phosphatase, Zellulase und Amylase Die Genomsequenzen ergänzen das genomischen Wissen über Vertreter der Gattung Spirosoma. 8 | S e i t e CHAPTER 1. Introduction and Background 9 | S e i t e 1. Introduction and background Climate change plays a role in increasing environmental stresses such as drought, salinization, floods and extreme temperatures that lead to reduction in crop productivity (Mahajan and Tuteja, 2005). Among them, soil salinity is one of the major threats to crops quality and productivity (Apse et al., 1999). Salinization has affected more than 20% of cultivated lands according to the Food and Agriculture organization (FAO) (Munns and Tester, 2008) and may increase with the accumulation of salt due to ongoing climate change (Corwin, 2020; Shrivastava and Kumar, 2015). The exponentially growing world population requires increasing food supply and hence soil remediation and salinity mitigation strategies are required to increase crop yields. 1.1 Salinity Salt stress is the excessive concentration of soluble salts such as NaCl (most common), Na2SO4, MgSO4, CaSO4, MgCl2, KCl, and Na2CO3 (Ilangumaran and Smith, 2017; Mukhopadhyay et al., 2020). A soil with an electrical conductivity (of the saturated paste extract) of 4 dS m-1 or more is considered a saline soil (Ilangumaran and Smith, 2017), equivalent to 40 mM NaCl and 0.2 MPa osmotic pressure (Munns and Tester, 2008). These conditions cause a significant reduction in the productivity of most crops. The source of dissolved salts in soils are physical or chemical weathering of primary rocks, geological depositions that releases salts, evapotranspiration phenomena from salt-affected groundwater that originates and deposits salts near surface, inflow of seawater or seawater flooding in coastal areas; these types of occurrences are also referred to as primary salinization (Daliakopoulos et al., 2016; Mustafa et al., 2019). Salts can also be introduced as a result of human activities such as land clearing, irrigation with saline water, cultivation activities, addition of chemical fertilizers, inadequate drainage; practices that are associated with secondary salinization (Daliakopoulos et al., 2016; Mustafa et al., 2019). Increasing climate change is negatively affecting the hydrological cycle, leading to a decrease in rainfall followed by prolonged droughts (Corwin, 2020). Thus, when rainfall levels are low to leach salt ions from the topsoil, salts accumulate, which is one of the main reasons for salinization (Akhtar, 2019). 10 | S e i t e 1.2 Effects of soil salinity on plant growth Plants are able to detect salt stress and trigger a series of changes at hormonal and physiological levels in response (Munns, 1992). This set of responses develops in two phases; an immediate phase known as the osmotic phase and an ionic phase that occurs after prolonged exposure to salt stress (several days or weeks) (Munns et al., 1995); both phases result in most vital processes in the plant being negatively affected, including, mineral transport, reproduction, photosynthesis and transpiration. The main consequence of osmotic phase is the loss of intracellular water, plant cells under normal conditions have higher osmotic potential than soil, which allows the uptake of water and nutrients from the soil solution, but under salt stress, the osmotic potential in the soil is higher, causing an osmotic imbalance, which impedes the plant from carrying out the normal uptake process from the soil (Passioura and Munns, 2000; Yeo et al., 1991). As an effect of the disturbed water balance, the synthesis of abscisic acid (ABA) is stimulated, whereupon the stomata are closed to prevent water loss. In addition, the closure of the stomata leads to the generation of reactive oxygen species (ROS) such as hydroxyl radicals, superoxides, disrupt cellular components and processes (Claeys et al., 2014; Pel et al., 2000; Wise and Naylor, 1987). Another consequence of osmotic shock is the reduction of photosynthesis rate, which leads to plant growth limitation as energy is expended to maintain ionic balance and water conservation (Allakhverdiev et al., 1999, 2000). On the other hand, the ionic phase is manifested by a constant accumulation of salt in the leaves, the ions of Na+ are transported to the leaves through the xylem, most of the ions remain in the leaves, but a small portion is returned through the phloem, causing Na+ to accumulate in the leaves, resulting in a mineral imbalance that leads to an interruption of leaf growth, a decrease in the photosynthetic process and senescence (Flowers and Yeo, 1986; Munns and Passioura, 1984). 11 | S e i t e 1.3 Plant growth-promoting rhizobacteria (PGPR) Soil bacteria are the most abundant group of microorganisms in soil with about 108-109 bacteria per gram of soil (Schoenborn et al., 2004). Most of them are found with a high population density in the rhizosphere zone of plant roots (DeAngelis et al., 2009), attracted by the presence of various metabolites such as amino acids, sugars and organic acids exuded by the plant (Zhalnina et al., 2018). A group of bacteria known as plant growth-promoting rhizobacteria (PGPR) are found in the rhizosphere and are of particular interest because of their beneficial relationship with plants; these bacteria have several mechanisms that promote plant growth, such as nutrient supply through biological nitrogen fixation (Marroquí et al., 2001)(Christiansen-Weniger and van Veen, 1991), phosphorus solubilization (Kumar et al., 2001; Rodriguez et al., 2004) siderophore production (Radzki et al., 2013), inhibition of phytopathogens agents (Mazurier et al., 2009), likewise, PGPR are able to produce or modulate phytohormones for plant growth, including indoleacetic acid (IAA), cytokinins, gibberellins and ethylene (Nieto and Frankenberger, 1989; Tien et al., 1979); they participate in the biodegradation of toxic organic compounds which promotes phytoremediation processes (Muratova et al., 2005; Weyens et al., 2015) 1.3.1 Alleviation of salt stress by PGPR PGPR have been reported to increase the tolerance of plants to environmental stress (Kumar et al., 2007; Ortiz et al., 2015; Staudinger et al., 2016) and especially in alleviating stress caused by salt in plants (Hmaeid et al., 2014; Porras-Soriano et al., 2009; Sukweenadhi et al., 2015) hence the application of halotolerant bacteria with PGPR activity on salt-affected soils is suggested to reactivate crop yields of plants. The mechanisms of PGPR conferring salt tolerance include the synthesis of cytokinins, indoleacetic acid, ACC deaminase, volatile organic compounds (VOC), exopolysaccharides, restoration of osmotic balance, regulation of oxidative stress, and modulation of gene expression (Forni et al., 2017; Ilangumaran and Smith, 2017). Plant growth-promoting rhizobacteria have been shown to be able to restore osmotic balance by modulating gene expression of proteins such as plasma 12 | S e i t e membrane aquaporin protein (ZmPIP), which is involved in stomata opening. Studies by Marulanda et al., 2010 showed that maize plants exposed to salt stress and inoculated with Bacillus megaterium increased the expression of ZmPIP, leading to a reduction in leaf’s damage and improvement of water conductance. The accumulation of osmolytes with osmoprotective properties maintains cell turgor and allows water to move from the soil into the roots; examples of osmolytes include proline, trehalose, and glycine betaines produced or induced by PGPR. Bacteria belonging to the genera Bacillus and Arthrobacter enhanced the accumulation of proline in maize plants under salinity conditions, thereby improving plant growth (Ullah and Bano, 2015), another study using soybean plants inoculated with Bacillus and Pseudomonas demonstrated an increase in proline concentration in roots, which allowed water uptake and improved shoot growth under salinity conditions (Kumari et al., 2015). Due to ion imbalance, plants have different mechanisms for Na+ efflux and K+ influx to diminish ion toxicity, one of which is the high-affinity potassium transporter (HKT), which increases the uptake of K+ over Na+ ions, PGPR can upregulate the expression of HKT genes in the roots and thus restrict the entry of Na+ into roots (Safdarian et al., 2019). Microbes also minimize the uptake of salt ions by producing exopolysaccharides that act as a physical barrier around the roots, trapping cations in their matrix to prevent uptake by plants (Akhtar, 2019). Another mechanism to restore osmotic balance regulated by PGPR is the release of volatile organic compounds (VOCs), these low molecular weight compounds are able to induce the production of ROS scavenger enzymes such as catalase, superoxide dismutase, glutathione reductase which protect the plant from cellular damage (Timmusk et al., 2014). Moreover, VOCs are found to upregulate the production of HKT proteins and subsequently promote growth and increase plant leaf biomass. HKT1 gene expression in roots and shoots is up-regulated in Arabidopsis thaliana in association with Bacillus subtilis under salt stress by exposure to VOCs, which induces a lower Na+ accretion (Zhang et al., 2008). Rhizobacteria favour the production of phytohormones, leading to the activation of various response mechanisms that help the plant cope with biotic and abiotic stress effects. 13 | S e i t e One of the most studied phytohormones produced by PGPR is indole-3-acetic acid (IAA) which is taken up by the plant and is involved in the stimulation of cell division (Khalid et al., 2004). The synthesis of IAA is given by the utilization of tryptophan, which is secreted by plants as a metabolite in the rhizosphere, which in turn is transformed to IAA by rhizobacteria (Dodd et al., 2010). During salt stress in tomato plants, IAA was shown to accumulate in the roots at a concentration of 100 mM NaCl (Albacete et al., 2008). However, once NaCl concentration is increased to 300 mM, IAA levels decrease significantly (Dunlap and Binzel, 1996). PGPR can alleviate salt stress in plants by producing IAA, shoot and root growth were stimulated by inoculating wheat with IAA-producing bacteria Pseudomonas aurantiaca and Pseudomonas extremorientalis at 100 mM NaCl (Egamberdieva, 2009). As described above, abscisic acid (ABA) is also induced under salt stress, ABA is induced in roots and then transported via xylem to leaves where it is involved in closing stomata to minimize transpiration activity and regulate water potential (Borel et al., 2001), PGPR are capable of producing ABA but their role in the plant- microbe interaction has not been elucidated, however, these microorganisms could enhance plant survival by regulating ABA pathways. The concentration of ABA in cucumber plants under salt stress inoculated with PGPR was reduced compared to plant controls, which improved the plant growth (Kang et al., 2014); in another report, ABA accumulation was reduced in cotton plants associated with Pseudomonas putida RS-198, which induced salt tolerance and increased plant biomass in salinized soil (Yao et al., 2010). Another important plant hormone associated with biotic and abiotic stress is ethylene, which is critical for seed dormancy breaking, rhizobial nodule formation, fruit ripening and leaf abscission, among others, but is also induced in high concentrations in response to stress phenomena that cause senescence (Glick, 2014). As a means to reduce stress and promote plant growth, PGPR have been shown to be able to inhibit ethylene biosynthesis by cleaving the precursor 1- aminocyclopropane-1-carboxylase (ACC) to α-ketobutyrate and ammonia with the release of the enzyme ACC-deaminase, thus reducing ethylene levels in plants (Glick, 2014); a study indicated the decrease in ethylene synthesis in red pepper plants grown at 150 mM NaCl and inoculated with ACC-deaminase-producing halotolerant bacterial strains Bacillus licheniformis RS656, Zihhengliuela alba RS111” and Brevibacterium iodinum RS16, resulting in an increase in nutrient 14 | S e i t e assimilation and increased tolerance to salt stress compared to non-inoculated control plants (Siddikee et al., 2011). Strains of Pseudomonas fluorescens and Enterobacter spp. containing ACC deaminase decreased induced ethylene levels and increased maize yield in salt-affected soils compared to the non-inoculated control (Nadeem et al., 2009). In conclusion, PGPR under salt stress induce systemic tolerance that promotes plant growth and development. Moreover, it is important to point out the enormous potential of microorganisms associated with the rhizosphere of halophytic plants or in the vicinity of saline soils to alleviate salt stress and promote crop plant growth. 1.4 Methylotrophs Methane and methanol are among the C1 compounds (one carbon-molecule) that are part of the carbon cycle and are widely distributed in nature (Iguchi et al., 2015). The production of methane occurs in anoxic environments by anaerobic archaea, also known as methanogenic archaea, which reduce CO2 with H2 (hydrogenotrophic) to CH4 or convert acetate to methane and CO2 (acetoclastic) and mostly depend of the activity of H2- and acetate-producing fermenting bacteria co-occurring in the same habitat. This group of microorganism can be found as free-living archaea in lakes, paddy fields (Maksimavičius and Roslev, 2020; Whiticar, 2020), as well as in specialized compartments such as the rumen of ruminants like cows (Vanwonterghem et al., 2016). Methane is also produced in abiotic processes such as coal mining, oil and gas operations, anaerobic waste treatment plants, landfills etc. (Iguchi et al., 2015; Whiticar, 2020). These processes have made methane the second most important gas produced by anthropogenic activities, and it is in turn part of the atmospheric gases that promote the greenhouse effect (Whiticar, 2020). On the other hand, methanol is a volatile carbon compound and the main contributors to its emissions are plants through the processes of repair and demethylation of pectin polymers in the cell wall as well as lignin breakdown (Fall and Benson, 1996; Galbally and Kirstine, 2002); however, other sources of methanol have been mentioned, including the chemical industry, where it is commonly used as a solvent (Fall and Benson, 1996), and other anthropogenic 15 | S e i t e activities (Singh et al., 2000) and it is one of the most abundant volatile organic compounds in the atmosphere (Galbally and Kirstine, 2002). Methane and methanol, as well as other C1 compounds, are used by methylotrophic organisms as the sole source of energy; furthermore, facultative methylotrophic organisms capable of utilizing multicarbon compounds have been identified (Chistoserdova et al., 2009; Nazaries et al., 2013); methylotrophs are widely distributed in ecosystems such as geothermal reservoirs, soils, peat bogs or aquatic environments and sediments, where most of them grow under oxic conditions at neutral pH (6.0–8.0) and temperatures between 10 and 40 °C (Nazaries et al., 2013), but acidic, alkaliphilic, thermophilic and psychrophilic species have also been described (Op den Camp et al., 2009); they are also representative members of Proteobacteria, Verrucomicrobia, Flavobacteria and Actinobacteria (Kolb, 2009; Stacheter et al., 2013). As mentioned above, plants can produce methanol as result of their biochemical activities, which can be exuded from the leaves or the roots, whereby phyllosphere (Stacheter et al., 2013) and the rhizosphere (Macey et al., 2020) have been described as zones with high densities of methylotrophs. Moreover, the presence of proteins involved in methylotrophic metabolism has been demonstrated in the phyllosphere and roots of rice plants (Knief et al., 2012) and of taxonomic groups with methylotrophic activity in wheat roots (Turner et al., 2013). Activities to promote plant growth under abiotic stress have been reported for methylotrophic bacteria relieving the adverse effects through activities such as increased uptake of essential elements through nitrogen fixation, P, K and Zn solubilization, production of chelating compounds, increase in tolerance through production of phytohormones (Kumar et al., 2019). 1.4.1 Aerobic oxidation of methane to formaldehyde Methanotrophic bacteria oxidize methane under oxic conditions via methane monooxygenase and dehydrogenases, producing intermediates such as methanol, formaldehyde, and formate, which are further converted to CO2 (Jiang et al., 2010; Macey et al., 2020). To catalyze the oxidation of methane to methanol, methanotrophic bacteria can produce two forms of methane monooxygenases (MMO) enzymes with the same metabolic function; particulate (pMMO) or soluble 16 | S e i t e methane monooxygenases (sMMO). The pMMO is a membrane-bound enzyme with copper in its active site, requiring a copper-sufficient environment for its functionality, the pMMO is the most common enzyme among methylotrophic bacteria; the sMMO, on the other hand, is a cytoplasmic enzyme with a di-iron active site that is released at low copper concentrations and is found in selected groups of methylotrophs (Jiang et al., 2010; Park and Lee, 2013). The methanol produced is subsequently oxidized to formaldehyde by means of a periplasmic pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH), encoded by the calcium dependent mxaF-gene or the xoxF-gene, the latter recently discovered to have lanthanide dependent activity; then the formaldehyde is converted to formate via tetrahydrofolate or tetrahydromethanopterin intermediaries, the pathway ends with the final conversion of formate to CO2 by NAD-dependent formate dehydrogenase. Alternatively, another part of formaldehyde can be assimilated into the cell biomass via the assimilatory ribulose monophosphate (aRuMP) pathway or via the serine cycle pathway (Figure. 1) (Kalyuzhnaya, 2016; Nazaries et al., 2013) Figure 1. Aerobic methane metabolism; membrane-bound methane monooxygenase (pMMO); soluble methane monooxygenase (sMMO); PQQ-linked methanol dehydrogenases (Mxa); PQQ-linked methanol and formaldehyde dehydrogenases (Xox); methanopterin-linked C1 transfer (H4MTP); folate-linked C1 transfer (H4FP); formate dehydrogenase (Fdh); NADH:quinone oxidoreductase (NDH2); assimilatory ribulose monophosphate pathway (RuMP); serine cycle (SC) (Modified from Kalyuzhnaya, 2016) The ribulose monophosphate pathway (RuMP pathway) occurs with the condensation of formaldehyde on ribulose 5-phosphate by 3-hexulose 6-phosphate synthase (Hps) to form hexulose 6-phosphate and its isomerization to fructose 6- phosphate by 3-hexulose 6-phosphate isomerase (Hpi). The fate of this six-carbon compound could occur via three predicted RuMP pathways, including the Embden- 17 | S e i t e Meyerhof-Parnas pathway (EMP) (glycolytic); the Entner-Doudoroff (EDD) pathway (KDPG variant); and the Bifidobacterium shunt (BS) variant (phosphoketolase, Xfp variant)( Figure. 2) (Kalyuzhnaya, 2016). Via all pathways important intermediates for biomass synthesis (sugars, acetyl-CoA, glycerin- phosphate) are provided. Figure 2. The ribulose monophosphate (RuMP) represented with the three predicted routes for cleavage for formaldehyde assimilation (a) EMP (FBA) variant; (b) EDD (KDPG) variant; and (c) BS (Xfp) variant. Dihydroxyacetone phosphate (DAP); 2,3- diphosphoglycerate (DPG); erythrose-4-phosphate (E4P); fructose 6-phosphate (F6P); fructose 1,6-bisphosphate (F1,6P); glyceraldehyde 3-phosphate (G3P); glucose 6- phosphate (G6P); 3-hexulose 6-phosphate (He6P); 2-keto-3-deoxy 6-phosphogluconate (KDPG); 6-phosphogluconate (6PG); phosphoenolpyruvate (PEP); 3-phosphoglycerate (PGA); ribulose 5-phosphate (Ru5P); ribose 5-phosphate (R5P); sedoheptulose 7- phosphate (S7P); (X5P) xylulose 5-phosphate; diphosphate (PPi); inorganic phosphate (Pi). (Modified from Kalyuzhnaya, 2016) The serine cycle, on the other hand, begins with the condensation of methylene- H4F and glycine to serine, which in turn, after several transformations, is converted to phosphoenolpyruvate, which is carboxylated to malate, followed by CoA- addition to generate malyl-CoA, which is cleaved to glyoxylate and acetyl-CoA, glyoxylate is finally converted back to glycine (Figure. 3) (Chistoserdova and Lidstrom, 2013). 18 | S e i t e Figure 3. The serine cycle in in methanotrophic bacteria. Methane monooxygenase (MMO); methanol dehydrogenase (MDH); methylene tetrahydromethanopterin pathway (H4MPTP); methylene tetrahydromethanopterin dehydrogenase (MtdA); formate dehydrogenase (FDH); serine hydroxymethyl transferase (STHM); hydroxypyruvate reductase (HPR); malate dehydrogenase (MD); malate thiokinase (MTK); malyl coenzyme A lyase (MCL). (Adapted from Fei et al., 2014) 1.4.2 Methanol dehydrogenase (MDH) The MDH is a protein found in the periplasm of most model methylotrophs; this protein has a prosthetic group (PQQ) placed in the center of a large α2 subunit folded in a β-propeller structure and a β2-subunit surrounding the α2- subunit (Figure 4). The mechanism of enzyme action begins when PQQ is reduced with a proton that is withdrawn from the alcohol group present in the methanol molecule by an active site dominated by Asp303 and a cofactor that acts as a Lewis acid (Anthony, 2004); this reduced PQQ is reoxidized transferring two electrons to the heme group of cytochrome CL, which in turn is oxidized by cytochrome CH. This latter compound transfers the electrons to a membrane-bound C-type, which could be cytochrome aa3 or CO (oxidase); the electron chain ends with a final step where the electrons reach the final acceptor oxygen (Anthony, 1992; Read et al., 1999), this process of electron transfer generates a proton motive force that can generate one molecule of ATP per molecule of oxidized methanol (Williams et al., 2005). 19 | S e i t e Figure 4. Structure of Methylobacterium extorquens MDH. The α-subunit folded in propeller-structure (green) coupled with a PQQ and a calcium ion (green sphere) in the active center, the small β-subunit (yellow) (Adapted from Williams et al., 2005). The first MDH was discovered in Methylobacterium extorquens, which is encoded by the mxaF-gene that requires calcium (Ca+2) as a cofactor for its activation; the mxa cluster in M. extorquens consists of the mxaF and mxaI genes, which express the large and small subunits of MDH, mxaG, which encodes cytochrome CL, mxaACKL which encodes proteins for calcium insertion, and other genes required for MDH activity (Chistoserdova, 2011). This MDH-Ca2+ dependence was considered the most traditional and widely used enzyme model for methanol oxidation in methylotrophic bacteria such as Methylobacterium sp., P. denitrificans, Hyphomicrobium methylovorum, and Methylophilus methylotrophus (Hibi et al., 2011a). Recently, however, Hibi et al., 2011 found an alternative MDH in Methylobacterium radiotolerans encoded by a xoxF-gene, a homologous gene of mxaF that is dependent on lanthanum (Ln3+) as a cofactor and widely predominant in methylotrophic bacteria and co-occurs with the mxaF gene or alone (Lv et al., 2018). The operon of XoxF includes the xoxF-gene, encoding Ln-dependent MDH, the xoxG-gene, encoding cytochrome CL and the xoxJ-gene, encoding a protein for La3+ transport (Featherston and Cotruvo, 2021; Keltjens et al., 2014). MDH encoded by xoxF shows optimal activity at neutral pH and is not dependent on ammonium, in contrast to mxaF, which requires pH around 9-10 and is dependent 20 | S e i t e on ammonium for its activity (Pol et al., 2014). Some of methylotrophic bacteria with xoxF activity include, Methylobacterium radiotolerans, Bradyrhizobium sp., “Candidatus Methylacidiphilum fumariolicum SolV”, M. extorquens AM1, and others (Hibi et al., 2011a; Pol et al., 2014). The rare earths elements (REEs) comprise a group of 17 chemical elements from lanthanum to lutetium with atomic numbers 57 to 71 in the periodic table and also include yttrium and scandium; rare earths elements are widely distributed as mixtures in the Earth’s crust and are classified into two groups according to electron configuration, mineralogy and chemical behavior: Light rare earths (LREEs), which include from La to Eu, and heavy rare earths (HREEs), which include Gd-Lu and Y (Hoshino et al., 2016). The rare earth elements (REEs) are used as components of electronic devices such as mobile-phones, solar cells, and computers (Pol et al., 2014) and are often found in insoluble forms such as oxides, silicates, carbonates, and phosphates (Habashi, 2013). Due to their low solubility, unestablished association with biological functions and mechanisms, these REEs were usually considered nonessential until their role as cofactors of MDH for methanol oxidation in methylotrophic species was discovered (Pol et al., 2014). The association of REEs coupled to PQQ exhibit a stronger Lewis character, which plays an important purpose in polarizing atoms and enhances the redox activity of MDH compared to Ca2+ (Bogart et al., 2015; Pol et al., 2014). It is evident that REEs are assimilated as described above, but, the mechanisms have not been elucidated, however, despite their low solubility, some evidence for the assimilation of these elements has been suggested. The presence of binding groups for REEs in the bacterial cell surface such as carboxylates and phosphates has been pointed out (Takahashi et al., 2005), another study on REEs uptake mechanism described the involvement of molecules with similar activity to chalkophores or siderophores involved in copper or iron acquisition, these compounds are known as lanthanophores which solubilize La3+ from mineral complexes, the lanthanophores are transferred to the periplasm by a TonB- dependent receptor in the outer membrane, once in the periplasm the La3+ is released from the lanthanophore and the La3+ is recognized by apo-xoxF or a periplasm binding protein that induces the expression of the xoxF cluster; another pathway involves the transfer of La3+ from the periplasm to the cytoplasm through 21 | S e i t e a ABC transport system coupled to the internal membrane; in the cytoplasm, La3+ also activates transcription of the xoxF cluster (Ochsner et al., 2019). 1.4.3 Expression Regulation of xoxF The gene XoxF is involved in carbon metabolism and is expressed when lanthanide species such as La3+, Ce3+, Pr3+and Nd3+ are available, but it prefers La3+, as shown by an experiment in which Methylorubrum extorquens strain AM1, growing in a mineral medium containing a mixture of Ln-species, used La3+ as a preferred cofactor (Xu et al., 2007). As mentioned above, methylotrophic bacteria can harbor both MDH-genes (mxaF-xoxF) and under growth conditions in an environment with methanol and free of lanthanides, normally express mxaF but preferentially induce xoxF at low concentrations of lanthanide elements, even when the Ca2+ concentration is higher. This mechanism is known as the “lanthan-switch”, in which transcription of the mxaF gene is repressed and expression of the xoxF gene is up- regulated in the presence of lanthanides (Chu and Lidstrom, 2016; Vu et al., 2016). A periplasmic binding protein has been identified in M. extorquens, that may be associated with the “lanthan-switch”. This is the La3+-selective chelator called Lanmodulin (LanM), which occurs in the periplasm and contains four carboxylate-rich metal coordination motifs known as EF hands, allowing it to selectively recognize La3+ ions. Depending on the organism, expression regulation may vary. Three-component systems, MxbDM, MxcQE, MxaB are involved in the regulatory mechanism of M. extorquens, but only the MxbM component is required for repression of the xoxF cluster in the absence of lanthanum, however, XoxF is required for expression of the mxa cluster and the MxbDM component system leads to repression of itself (Featherston and Cotruvo, 2021; Vu et al., 2016); to fulfill the requirement of xoxF for expression of the mxa cluster, Skovran et al., 2019 propose the involvement of apo-xoxF (metal not incorporated into xoxF) as a lanthanide sensor in periplasm that interacts with either MxcQ or MxbD; once apo-xoxF reacts with La3+, it triggers transcription of the xoxF operon and represses mxa expression. In another methylotrophic bacteria Methylomicrobium buryatense, the component system of MxbDM and MxcQE is not present, but a sensor kinase known as MxaY, which can recognize Ln3+, regulates the expression of mxa and xoxF clusters (Chu et al., 2016). 22 | S e i t e 1.5 Hartmannibacter diazotrophicus E19T Hartmannibacter diazotrophicus E19T is a gram-negative bacterium isolated from the rhizosphere of the plant Plantago winteri Wirtg., as part of bacterial screening project with PGPR potential associated with halotolerant plants growing in a natural salt meadow. Taxonomically, the bacterium belongs to the Domain Bacteria, Phylum Proteobacteria, Class Alphaproteobacteria (Suarez et al., 2014) and was recently assigned to the Order Hyphomicrobiales, novel family Pleomorphomonadaceae along with other genera such as Chthonobacter, Methylobrevis, Mongoliimonas, Oharaeibacter, and Pleomorphomonas (Hördt et al., 2020). H. diazotrophicus is described as motile, strictly aerobic, oxidase-positive and catalase-negative, grows in 1–3% (w/v) NaCl, the major respiratory quinone is Q- 10, DNA G+C content is 59.9% ± 0.7 mol%. The activities described to promote plant growth of H. diazotrophicus include: Phosphate solubilization, nitrogen fixation, ACC and VOC production, iron uptake. The ability of phosphate solubilisation by H. diazotrophicus was tested in liquid medium with inorganic phosphorus complex sources [Ca3(PO4)2, AlPO4 and FePO4] and organic ones such as inositol hexaphosphate (IHP), obtaining values ranging from 0.15 to 0.37 mg·l- 1 of soluble phosphate; it was also observed that gluconic acid is the most produced organic acid for solubilization activity (Suarez et al., 2019). The gluconic acid is synthetized in the oxidation pathway of glucose by means of enzyme glucose dehydrogenase and a cofactor pyrroloquinoline quinone (PQQ) (Suleman et al., 2018). The gene pqqBCDE, encoding the biosynthesis of PQQ and a substitute of the enzyme glucose dehydrogenase encoded by yliI are present in the H. diazotrophicus genome, but genes for acidic phosphatase, alkaline phosphatase and phosphatidic acid production are also present (Suarez et al., 2019). Nitrogen fixation by H. diazotrophicus was confirmed by its growth on nitrogen-free medium and nitrogenase activity by acetylene reduction to ethylene; genes involved in the biological nitrogen cycle were annotated in its genome. The genes described for nitrogen fixation activity include nifH, nifD and nifK, which encode the structural subunits of nitrogenase, genes involved in the synthesis and incorporation of FeMoco into nitrogenase, such as nifE, nifN, nifX, nifB, nifQ and nifV, as well as other genes involved in the protection, stabilization and regulation of nitrogenase 23 | S e i t e (Suarez et al., 2019). Mechanisms for the acquisition and metabolism of sulfur and iron in H. diazotrophicus are possible through specific protein complexes in the outer membrane; mixed sulfur compounds, e.g. alkane sulfonates, are taken up by an aliphatic sulphonate ABC transport encoded by ssuABC; once inside the cell, sulfur compounds are mineralized to sulfite by an alkane sulfonate monooxygenase and an NADPH-dependent FMN reductase encoded by the genes ssuD and ssuE genes respectively. Second, the iron is chelated by a siderophore, which in turn is transported by the protein complex of TonB, ExbB and ExbD into the periplasmic space, where the siderophore releases the ferrous ion and the iron is taken up by a Fe-binding protein and transported to the cytoplasm via an ATP transporter. It has been mentioned that ACC-deaminase can degrade the precursor 1- aminocyclopropane-1-carboxylase (ACC), thereby lowering the ethylene levels in plants while reducing stress from salt (Glick, 2014), H. diazotrophicus was able to grow in a ACC-supplemented medium (Suarez et al., 2014), moreover, genes with enzymatic potential for ACC-degradation are present in its genome (Suarez et al., 2019). A metabolic pathway for the production of VOCs is also evidenced with the gene ilv, which encodes the enzyme acetolactate synthase for acetolactate production, and the gene budC, which encodes diacetyl reductase that subsequently converts acetolactate to acetoin by decarboxylation. H. diazotrophicus possesses genes for proline and glutamate biosynthesis, the genes otsA and otsB, which express the production of trehalose synthase and trehalose phosphatase, involved in the synthesis of trehalose, and the genes betA, betB, which encode for choline dehydrogenase and betaine aldehyde dehydrogenase, essential enzymes for betaine synthesis (Suarez et al., 2019). Trehalose, proline, glutamate, betaine belong to the group of compatible solutes that plants accumulate as osmoprotectants during salt stress (Ullah and Bano, 2015). Other gene-activities identified in H. diazotrophicus that may help the plant to cope with salt stress are peroxidases, superoxidase and glutathione S-transferase, which are encoded in coding sequences (CDS) and are involved in the protection of the cell against oxidative stress, also the KDP operon, which is involved in the expression of high-affinity K+ and favors the uptake of K+ over Na+, and the gene cluster mrpABCDEFG, which stimulates Na+ efflux and regulates cell volume and pH homeostasis (Suarez et al., 2019). In methylotrophic bacteria, two genes have been associated with methanol dehydrogenase expression, mxaF and xoxF (Daumann, 2019). In H. 24 | S e i t e diazotrophicus, the presence of the xoxF4-lanthanide-dependent gene was detected and its activity was confirmed by growth of the bacteria in mineral medium supplemented with methanol and lanthanum (Lv et al., 2017). The genome of H. diazotrophicus harbors genes for other methylotrophy activities associated such as PQQ synthesis, H4MPT and H4F pathways, formate oxidation, serine cycle and methylamine dehydrogenase (Lv and Tani, 2018; Suarez et al., 2019). The plant growth-promoting activity of H. diazotrophicus in planta was demonstrated when barley plants growing under salt stress were inoculated, which increased shoot and root dry weights compared to non-inoculated plants; moreover, ethylene levels were reduced as a possible consequence of ACC deaminase activity (Suarez et al., 2015); another study showed that inoculation of alfalfa plants with H. diazotrophicus under salinity enhanced K+ uptake and carotenoids production and increased relative water content (Ansari et al., 2019). 1.6 Barley Barley (Hordeum vulgare, vulgare L.) is a cereal crop which grows in a variety of climates from sub-Arctic to subtropical and is listed on position five of crops with the highest dry weight production in the world. Barley is composed primarily of soluble and insoluble dietary fiber, with β-glucans predominating, but is also a source of protein, carbohydrates, vitamin E, B-complex vitamins, minerals, and phenolic compounds. Barley is incorporated in breakfast cereals, soups, baking flour and is an important food source that is also used in the production of alcoholic beverages such as beer (Gupta et al., 2010). Barley belongs to the grass family Poaceae, subfamily Festucoideae, tribe Triticeae and genus Hordeum. The tribe Triticeae includes the best known cereals, namely: wheat (Triticum , several species), rye (Secale cereale), barley (H. vulgare) and others (Von Bothmer and Komatsuda, 2011). Of genus Hordeum, thirty-one species have been described (Von Bothmer and Komatsuda, 2011) but the cultivated barley varieties belongs to the species: vulgare and the wild form which belongs to the species spontaneum. Morphologically, barley is similar to other grasses and has spikes consisting of a series of spikelets which in turn contain 25 | S e i t e florets, the organization of the spikelets can be two-rowed or six-rowed, resulting in differences in composition and reproduction (MacGregor, 2003). 1.7 Taxonomical description of a new Spirosoma species The study of the biodiversity and relationships among living organisms is facilitated by taxonomical classification (Gevers et al., 2005); for bacteria, the taxonomy enables their reliable identification from clinical and environmental samples (Moore et al., 2010). Currently, a polyphasic approach is the standard method for bacterial novel taxa classification (Tindall et al., 2010), which consists of a series of tests for the evaluation of phenotypic, genotypic, and chemotaxonomic traits (Stackebrandt et al., 2002). Phenotypic tests comprises morphological, physiological, and biochemical characteristics (Wayne et al., 1987); genotypic methods are referred to DNA-based analysis such as %G+C content, DNA-DNA hybridization (DDH) and 16S rRNA gene sequence analysis (Stackebrandt and Ebers, 2006; Tindall et al., 2010), and chemotaxonomic characterization on the other hand, include tests to evaluate differences in the structural components such as the cell wall, the cell membrane or cytoplasm (Tindall et al., 2010). In bacterial taxonomy, the species is the basic unit of bacterial taxonomy (Wayne et al., 1987) and can cluster similar strains which share 70% or more DNA-DNA relatedness. For the delineation of new species multiple rules of thumb have been proposed such as, DDH value which calculate genetic similarities at genome level between two species, value that should be less 70% to be designated as different species (Wayne et al., 1987); comparative sequence analysis of the 16S rRNA gene with identity values < 98,7% (Stackebrandt and Ebers, 2006) in combination with tests for evaluation of chemotaxonomy, physiological and cultural characteristics. The genus Spirosoma was described first time by Migula, 1894 and is assigned to the proposed family Spirosomaceae, phylum Bacteroidetes, the family description was emended by García-López et al., 2019. The genus comprises 42 members validly published (Euzéby, 2018) that were isolated from natural environments such as water, soil, dust and soil (Lee, et al., 2018; Ten, et al., 2018). Members of the genus are described as Gram-negative, aerobic or facultatively anaerobic, non-spore-forming, variable motility, rods with various morphologies such as 26 | S e i t e rings, coils, filaments, colonies pink to yellow pigmented, the major menaquinone is MK-7 (Ahn et al., 2014), phosphatidylethanolamine as major polar lipid and G+C content from 47.2 to 57.0 mol% (Chang et al., 2014). 1.8 Comparative genomics Genome sequencing is a tool of valuable importance, as it can aid in the taxonomic classification, the study of dynamics, evolution, and function of an unknown organism in an ecosystem. (Andersson and Goodman, 2012; Loman and Pallen, 2015) The use of NGS (Next generation sequencing) technologies in combination with informatic tools enable to sequence any bacterial genome project. A downstream analysis for a genome sequence involves the assembly of individual sequence reads into larger contigs; gene annotation and functional prediction based on referenced sequence databases to check genetic and metabolic capabilities of the bacteria; and comparative sequence analysis of single genes or whole genomes to genotypic characterization (Del Chierico et al., 2015; Roumpeka et al., 2017), core and even pan-genome analysis for multiples strains of species (Bentley, 2009). 1.9 Aim of the study Soil salinization negatively affects plant growth and all their metabolic processes and is also one of the major challenges for agriculture due to increasing climate change (Mukhopadhyay et al., 2020). PGPRs have been shown to alleviate the negative effects caused by salt stress in plants through ACC deaminase, phytohormones and VOCs production (Etesami and Maheshwari, 2018). Among PGPRs, salt halotolerant bacteria were found to have activities as plant growth promoters eg. H. diazotrophicus which showed improved growth of barley and alfalfa under saline conditions (Ansari et al., 2019; Suarez et al., 2015), however, the gene xoxF-lanthanide-dependent was amplified in H. diazotrophicus which confers the ability to methylotrophy through methanol uptake (Lv et al., 2017). The study of lanthanide dependent methylotrophy has attracted particular interest in recent years and most studies on methylotrophic bacteria with xoxF activity associated with plants have been conducted in the phyllosphere, but there is little 27 | S e i t e information on xoxF-methylotrophic bacteria with plant-promoting growth activity associated with roots, apart from a few studies on halotolerant bacteria dependent on lanthanides. Since methanol is a by-product of cell wall metabolism in plants, it can be released from either leaves and shoots (Macey et al., 2020; Stacheter et al., 2013), which could allow methylotrophic metabolism to influence plant-associated microbes and benefit the plant as PGPRs. In this study a greenhouse experiment with barley growing in saline soil was carried out, several treatments were established where plant seeds grow with/ without bacteria inoculation, with/without lanthanum supplementation, non- inoculated seeds grown in presence/absence of lanthanum and the controls of seeds only growing in saline soil without further amendments. The aims of this study were as follow: (I) Reassess the growth of H. diazotrophicus with methanol supported by lanthanum and to find the optimal concentration required as a metal cofactor for methanol dehydrogenase. 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Effect of Hartmannibacter diazotrophicus and lanthanum on the plant growth and microbial communities of barley grown under salt stress Submitted to: Biology and Fertility of Soil 44 | S e i t e Effect of Hartmannibacter diazotrophicus and lanthanum on the plant growth and microbial communities of barley grown under salt stress Julian Rojas1; Christian Suarez1; Stefan Ratering1; Marina Krutych1; Angelina Hormaza2; Sylvia Schnell1* 1Institute of Applied Microbiology, IFZ, Justus-Liebig University Giessen, 35392 Giessen, Germany 2 Faculty of Science, Universidad Nacional de Colombia - Sede Medellín, 050034 Medellín, Colombia *Author for correspondence: Sylvia Schnell E-mail: sylvia.schnell@umwelt.uni-giessen.de Abstract Increasing agriculture soil salinity caused by intensive fertilization or deficient irrigation negatively affects agricultural crop production. Hartmannibacter diazotrophicus E19T was isolated from a salt meadow and exhibited plant growth promoting activities. Substrates provided from plants like methanol support associated microorganism and genome analysis of H. diazotrophicus identified the gene xoxF which encodes for methanol dehydrogenase protein and confers the ability to grow on methanol only in the presence of lanthanum (La). For elucidation of lanthanum on plant growth promotion effects of H. diazotrophicus on barley under salt stress conditions (NaCl 3%) seed inoculation was performed in the presence of La. For a greenhouse experiment soil was amended with and without La (8 mg kg-1) and barley seeds were inoculated with alive or dead H. diazotrophicus. Plant growth, H. diazotrophicus colonization and bacterial community composition and diversity of rhizosphere and roots were studied. Significant differences of fresh and dry weight biomass of leaves and roots were obtained with H. diazotrophicus in the presence and absence of La3+. Treatments with La amended soil (without bacterial inoculation) also evidenced significant differences in leaves and roots biomass under salt stress compared to dead biomass addition of the respective bacterial strain and control. Presence of H. diazotrophicus in roots was 4.3 106 target copies gdw-1 as quantified by realtime PCR using a specific primer set for H. diazotrophicus based on the 16S rRNA gene. Similar target numbers were obtained with a primer set for the La-dependent methanol dehydrogenease gene (xoxF). Bacterial community analysis on roots and rhizosphere revealed Rhodanobacteriaceae (29.3%) as most dominant together with other families such as Acidobacteriaceae and Caulobacteraceae. Interestingly the alpha diversity analysis indicated higher indices in treatments with H. diazotrophicus, and in presence of La. According to nonmetric multidimensional scaling (NMDS) most similar communities were found in treatments of H. diazotrophicus and H. diazotrophicus with La; most different was the community in control soil. Differential abundance analysis showed Rhodanobacter as most frequent ranked genus in all treatments 45 | S e i t e This study reasserts the role of H. diazotrophicus in growth promotion of barley plants under salt stress and the importance of lanthanum for methylotrophic bacteria xoxF4-type. Keywords: Hartmannibacter diazotrophicus, Salt stress, PGPR, Lanthanum, Methylotrophic Bacterium, XoxF Introduction Salinization is a problem which affects according to FAO (United Nations Food and Agriculture Organization) already 22% of agricultural lands (Kolodyazhnaya et al., 2009). As a result of the greenhouse effect this value is increasing constantly due to low precipitation and the need of more irrigation. It has been predicted, that more than 50% of the cropland might be salinized by the year 2050 (Ashraf, 1994; Vinocur and Altman, 2005). The soil salinity has a negative impact in agricultural land decreasing crop yield. High concentration of salt ions such as Na+, K+, Cl-, Ca+ lead to osmotic stress and inhibits plant growth (Numan et al., 2018; Shrivastava and Kumar, 2015). The constant growing of world population requires to develop strategies for soil recovering and mitigation of salinity for a further increase of crop yield. One strategy to alleviate salinity stress for crop plants is the application of plant growth promoting rhizobacteria (PGPR) which can be inoculated to crop seeds and by different mechanisms enable better plant growth in saline soil. These mechanism include induction of systemic tolerance (IST), production of ACC-deaminase, support with nutrient uptake and resistance to phytopathogens (Suarez et al., 2015; Yang et al., 2009; Zarea et al., 2012). Hartmannibacter diazotrophicus E19T was isolated from a salt meadow and was shown to promote barley growth under salt stress (Suarez et al., 2015). Recently the genome of H. diazotrophicus was sequenced and candidate genes for potential plant growth promoting activities were recognized (Suarez et al. 2019) such as nif genes involved in nitrogen fixation, genes for acid and alkaline phosphatase associated to phosphorus metabolism, genes for protein expression of tonB, exbB, and exbD related to iron acquisition and also board clusters of genes involved in salt tolerance. Also three CDSs encoding for methanol dehydrogenase genes were found (Suarez et al. 2019) that as previously shown (Lv and Tani, 2018) belongs to xoxF type of the methanol dehydrogenases, that required for its activity the presence of rare metals as a cofactors such as lanthanum (La3+) and Cesium (Ce+3) (Chu and Lidstrom, 2016; Fitriyanto et al., 2011; Lv et al., 2017; Pol et al., 2014). H. diazotrophicus expressed only this type and cannot oxidize methanol without lanthanides (Lv et al., 2017). Methanol is a byproduct of pectine demethylation during the process of cell walls maturation in plants which is exudates in leaves (Fall and Benson, 1996; Galbally and Kirstine, 2002) and was also suggested in roots (Sy et al., 2005). In the root zone and below the xoxF type methanol dehydrogenases was found to be one of the most abundant expressed bacterial proteins (Butterfield et al. 2016, Li et al. 2019) and thus methanol could be an important carbon source of H. diazotrophicus on the root surface. In this work we tested the following working hypothesis: (I) Performance of H. diazotrophicus as plant growth promoting rhizobacterium on barley plants under salt stress is enhanced in presence of lanthanum. (II) Presence of lanthanum without seed inoculation with H. diazotrophicus has no effect on 46 | S e i t e plant growth. (III) Availability of lanthanum improves rhizosphere abundance of H. diazotrophicus. (IV) Bacterial communities are affected by strain E19T inoculation and lanthanum application in rhizosphere and roots. For testing our hypothesis, a greenhouse experiment was performed with barley under saline soil conditions and soil amendments with/without lanthanum and with/without seed inoculation of barley. Plant biomass, rhizosphere abundance of H. diazotrophicus, and effect of amendments of rhizosphere bacterial community were determined. Material and Methods Hartmannibacter diazotrophicus E19T was recultured from a glycerin stock from the culture collection of the Institute of Applied Microbiology, JLU Giessen. Growth of H. diazotrophicus on methanol For confirmation of lanthanum dependent methanol growth of strain E19T of our stock culture, a growth test was performed to proof enzymatic activity of the La-containing methanol dehydrogenase of H. diazotrophicus. A modified liquid freshwater media (FWM) (Widdel and Bak, 1992), containing 1.0 g NaCl, 0.4 g MgCl x 6H2O, 0.15 g CaCl2 x 2H2O, 0.5 g KCl, 0.2 g KH2PO4, 0.25 g NH4Cl, 1.4 g Na2SO4, 1.0 ml trace element solution containing no lanthanum (SL10), 50 ml Phosphate buffer 0.4 M pH 7.0, 1.0 ml vitamin B12-solution, 1.0 ml vitamin-5 solution, 1.0 ml thiamine solution, 1.0 ml riboflavine solution in 1.0 liter deionized water, the pH was adjusted to 6.5. Methanol 5% v/v as carbon source and 30 µM LaCl3 was added to this liquid culture medium, as well as H. diazotrophicus as inoculum which has been grown on glucose. As further growth tests 5 mM glucose (as a positive control of growth), no carbon source and methanol without lanthanum were included. The cultures were incubated at 28 °C on a shaker with 120 rpm. Growth was monitored by measurements of optical density at 600 nm. Another growth experiment was performed with different concentrations of LaCl3 (0, 30,60,90,120 µM), methanol 5% v/v under slightly saline conditions with 1% NaCl in the above described medium in order to test the optimal lanthanum concentration for growth. Growth experiment with barley in presence of H. diazotrophicus The effect of H. diazotrophicus E19T on growth of barley seeds was studied in pots filled with soil substrate under salt stress (NaCl 3%). Pots were arranged in a randomized complete block design (RCBD; McKillup, 2011), with six treatments and five replications per treatment. The treatments were (I) E19T, (II) E19T with La, (III) E19T dead biomass, (IV) E19T dead biomass with La, and (V) non-inoculated seed control. Two suspensions of H. diazotrophicus were obtained by growing the bacteria in FWM liquid medium with glucose or FWM with addition of methanol and 30 µM LaCl3 until 107 CFU ml-1. The suspension was centrifuged and suspended in 30 mM MgSO4. Seeds of barley were disinfected with 70% ethanol, 2.5% bleach, washed five times with sterile water and incubated for 1 h in the suspension of H. diazotrophicus grown in FWM with glucose for treatments of strain E19T without La or FWM with 47 | S e i t e methanol for treatments of strain E19T with La with gentle shaking. Further control assays consisted of barley seeds inoculated with an autoclaved aliquot of H. diazotrophicus E19T to assess the effect of dead bacteria and an assay with non-inoculated seeds. Plastics pots (13 x 10 cm) were filled with ~ 750 ml (~140 g dry weight) non-sterile soil (Fruhstorfer Erde Hawita typ P, Table. S1). Soil water holding capacity (WHC) was determinate to be 225 ml and each pot was watered with 170 ml water (~75% WHC) in order to keep the soil substrate moistened. Ten barley seeds were placed on the filled soil pots and were covered with 1 cm layer of soil substrate. The experiments were carried out in a greenhouse with 12 h of light, 21 °C of temperature. After germination the pots were rarefied to 6 plants per pot. In order to establish salt stress, the pots were irrigated three times during sowing, germination and rarefaction phases with 136 mM NaCl solutions in water until a salinity of 3.0% was reached. To evaluate the sorption of La3+ for the used substrate, the substrate without plants was flushed with solutions containing different La3+ concentration and the concentration of La3+ was determined by ICP-MS analysis in the runout (Table S2). Afterwards for the plant experiment a solution LaCl3 in water was added into the soil during seeding to obtain a concentration of 8 mg La kg-1. Plants were monitored for 35 day. Five plants per pot were separated for harvest of leaves and roots and collected in paper bags for determination of fresh weights. From root biomass soil was washed out thoroughly and air dried before fresh weights were determined. For dry biomass leaves and roots were dried at 80 °C for 48 h and subsequently their weights were recorded. Data normality was evaluated by Anderson-darling-test (Fig. S3); statistical differences of plants fresh and dry weight between treatments were analyzed by ANOVA followed by Tukey post-hoc tests at P<0.05 using the Minitab 19 Statistical Software (2019). DNA extraction from rhizosphere and roots From each pot one plant was separated and carefully the soil loosely adhering to the roots was removed by gentle shaking, collected in sterile centrifuge tubes and frozen to -80 °C. Additionally from the same plant one long root was cut out, remaining soil was removed by gentle shaking, afterwards root was collected in DNA-free tube and stored at -80 °C. For DNA extraction samples of rhizosphere were defrosted and kept in ice box. Approximately, 300 mg rhizosphere soil was transferred to tubes with ceramic beads and DNA was extracted using NucleoSpin ® Soil kit (Macherey- Nagel, Düren, Germany). Roots samples were disrupted to fine powder with a mortar and pestle using liquid nitrogen. Mortar and pestle were before use washed with soap, then rinsed with deionized water, flame treated with 100% ethanol and in last step the tools were wiped with ROTI®Nucleic Acid-free solution (ROTH, Germany). From the powder 300 mg was transferred to tubes with ceramics beads. To these tubes with powdered roots and ceramic beads 1 ml extraction buffer (5 g l-1 SDS, 0.2 M sodium phosphate buffer (pH 8), 50 mM EDTA and 0.1 M NaCl, pH 8) was added and shaken for 2 min at maximum speed in a cell mill MM200 (Retsch Haan, Germany). Then samples were centrifuged at 4 °C and 10.000 g for 5 min in a microcentrifuge (Heraeus Fresco, Thermo Fisher Scientific Inc., Waltham, USA). The supernatant was collected in a new tube, added 5 µl RNase A (10 mg ml-1) and incubated at 37 °C for 30 min. 48 | S e i t e DNA from roots was extracted using a modified phenol-chloroform methodology (Bürgmann et al., 2001). After RNAse A treatment 500 µl of phenol/chloroform/isoamyl alcohol (25:24:1) were added and mixed by inverting the tubes 5 times. The tubes were centrifuged again at 10.000 g for 5 min at 4 °C; aqueous phase was collected in a new DNA-free tube and chloroform/isoamyl alcohol (24:1) was added, mixed by gentle inverting and centrifuged again at 10.000 g for 5 min at 4 °C. Aqueous phase was recovered and DNA was precipitated with 1 ml of PEG [20% Poly (ethylene glycol) and 2.5 M NaCl], tubes were kept in ice box at 4 °C for 30 min and centrifuged again at 10.000 g for 5 min at 4 °C; PEG was discarded and DNA pellets were washed with ice cold 75% ethanol, dried out, dissolved in nuclease free water. DNA extracts of rhizosphere and roots were used for quantification of strain E19T by real- time PCR and taxonomic metabarcode sequencing by Ion Torrent. Quantification of H. diazotrophicus by real-time-PCR (qPCR) A specific primer set (E19_F_932: 5’-GTCCGGCTATCCAGAGAGAT-3’; E19_R_1261: 5’- ATTAGCTGACCCTCGAGGGT-3’) targeting the 16S rRNA gene of H. diazotrophicus has been developed through alignment of 16S rRNA gene of strain E19T and the closest relatives. The sequences obtained from the SILVA database (Quast et al., 2013) were aligned and merged with the database LTPs111 (Feb 2013) (Yilmaz et al., 2014) using the ARB version 5.2 program (Ludwig et al., 2004). The specificity of the primer pair was checked using the online program Probe Check (Loy et al., 2008) and also by cloning and sequencing of the PCR products amplified from DNA from bulk and rhizospheric soil of plants in two different environments. Likewise, a primer pair set (E19_F_xoxF: 5’- CGCTGCCATCTCACTGCCTA-3’; E19_R_xoxF: 5’-ACTGGTCGCCTTCCCATGTG-3’) targeting the xoxF-gene in strain E19T was designed on primerBLAST tool of NCBI (Ye et al., 2012) using NZ_LT960614, region: 2806010-2807833 sequence of the GenBank; the specificity of the primers were confirmed as well with primerBLAST. For cloning, PCR products were cleaned using NucleoSpin gel PCR clean-up (Macherey-Nagel) and ligated by TA-cloning to a vector plasmid pGEM-T (Promega GmbH, Mannheim, Germany); competent cells of Escherichia coli JM 109 (Promega) were transformed with the plasmids thereafter plasmids from transformed cells were isolated as described in Kampmann et al., 2012. The purified plasmids with insert were quantified using the fluorescence of DNA-quantification Helixyte Green kit (Biomol, Hamburg, Germany), copy numbers of standard fragments were calculated according to Kampmann et al. (2012) and used as a standards for real-time PCR curve in 10-fold dilutions from 106 to 101 of strain E19T 16S rRNA gene copies. Real-time PCRs were carried out in 10-µl reaction mixture volumes using a 72-well rotor in a Rotor- Gene 3000 cycler (Corbett Research, Sydney, Australia). All samples including the standard curve were run in quadruplicates starting with a preheating step at 94 °C for 2 min followed by 40 cycles of thermal cycling protocol: 45 s at 94 °C, 45 s at 63 °C, 45 s at 72 °C and 15 s at 84 °C. After the last amplification step, a melting curve was obtained by heating the amplification product from 60 °C to 95 °C and SybrGreen fluorescent (SYBR-Green JumpStart Ready Mix, Sigma-Aldrich, St. Louis, USA) 49 | S e i t e measurement was performed with Qiagen Software Rotor Gene Q 2.3 (Qiagen, Hilden, Germany). The copy numbers were determined using the reference curve plotting the log of starting target amount versus threshold cycle, from the equation of reference DNA plots the initial concentration of each unknown sample was calculated. Data normality was tested using the method of Anderson-Darling and analysed by ANOVA with Tukey´s post-hoc at P<0.05 test in Minitab. Ion torrent sequencing For sequencing of rhizosphere and root bacterial community a procedure according to Kaplan et al., (2019) was followed; PCR reactions were performed for the amplification of the 16S rRNA gene using a general bacterial primer sets (520_F: 5’-AYTGGGYDTAAAGNG-3’; 926_R: 5’- CCGTCAATTYYTTTRAGTTT-3’) (Claesson et al., 2009; Engelbrektson et al., 2010) targeting hypervariable regions V4 and V5 of bacteria. Purified PCR products of samples were transferred to a 314 chip and a 318 chip (Ion PGM Hi-Q Sequencing kit , Ion 314v2 chip & Ion 318v2 chip) (Life Technologies) and sequenced using Ion PGM Sequencer (Life Technologies). Sequence data obtained were analyzed using the microbiome analysis package QIIME2 (version 2020.6) (Bolyen et al., 2019) . After importing the sequencing in the QIIME2 format the reads were demultiplexed with the tool cutadapt (Martin, 2011) then quality filtered, chimeras removed, clustered and dereplicate using DADA2 (Callahan et al., 2016). After removing sequences with plastid and mitochondrial origin, the dereplicated sequences were classified using a pre-trained Naive Bayes classifier (Pedregosa et al., 2011) trained with the SILVA 132 database (Quast et al., 2013). Alignment of the sequences were done with MAFFT (Katoh and Standley, 2013) and the phylogenetic tree using the maximum-likelihood algorithms was generate using FastTree (Price et al., 2010). Alpha diversity indexes were compared statistically using Minitab 19 and beta biodiversity analysis was compared statistically using the “vegan “ package within the R software environment version 3.6.3 (R Core Team, 2020) with R Studio version 1.3.1056 (RStudio Team, 2020). The alpha diversity indexes Observed ASVs and Shannon were calculated to estimate species diversity, normality data test was performed as aforementioned and ANOVA test along with Tukey at P<0.05 was used to analyze the effects of the treatments in roots and rhizosphere soil and their interaction with microbial alpha diversity. For beta diversity analysis a Non-metric multidimensional scaling (NMDS) was performed to reproduce the influence among the treatments in roots and rhizosphere soil on the Bray-Curtis dissimilarity matrix by applying the Adonis function permutational multivariate analysis of variance (ADONIS) (permutations = 999). Differential abundance testing was performed using the QIIME2-plugin Songbird (Morton et al., 2019) following the procedure described by Estaki et al. (2020). The method applies a multinomial regression model to estimate differential ranks of taxa across the treatments. 50 | S e i t e RESULTS Lanthanide-dependent methylotrophic activity of H. diazotrophicus Fresh water medium supplemented with 0.5% (v/v) methanol in presence of La3+ was used to test the growth of H. diazotrophicus using methanol as carbon source (Fig. S1). It is evident that the presence of La3+ promote growth of H. diazotrophicus and is required for growth on methanol as source of carbon. In addition, growth was evaluated under slightly saline conditions with 1% NaCl and different concentrations of lanthanum (30, 60, 90, 120 µM) and methanol 5% (v/v) to evaluate the optimal La3+ concentration. Results indicated that 60 µM La3+ was slightly the best concentration for growth of H. diazotrophicus (Fig.1). 1.0 La 0 0.8 La 30 La 60 0.6 La 90 La 120 0.4 0.2 0.0 0 2 4 6 8 10 Time (Days) Fig 1. Growth of H. diazotrophicus in mineral medium with NaCl 1%, 5% methanol (v/v) under different concentrations of La3+ (0, 30, 60, 90,120 µM). Values represent the average of triplicate replications with corresponding standard deviations, the optical density (OD600) was used as growth parameter. Plant growth promotion activity and colonization of H. diazotrophicus E19T on barley The effect of H. diazotrophicus E19T inoculation on fresh and dry weights of barley under salt stress conditions of greenhouse experiment was test after 35 days (Fig. 2), normality test on the data using the Anderson-Darling test revealed all of the data sets were normally distributed (Table. S3). Overall, the tendency is similar for fresh or dry weight of plants inoculated with strain E19T. A significant difference was revealed in fresh (F5,20=5.45, P=0.003), (F5,20=11.35, P<0.001) and dry weight (F5,20=57.78, P<0.001) (F5,20=15.91, P<0.001) of leaves and roots. The biomass was higher in plants which have been inoculated with strain E19T and also in presence of La3+ compared to treatments with dead biomass (DB), dead biomass with lanthanum (DB+La) and the corresponding control (non- inoculated plants). Plants inoculated with H. diazotrophicus showed an increase in leaves biomass by 5, 5 and 18% and in roots biomass by 38, 36 and 49% against DB, DB+La and the control (Tab. S4) (Tukey HSD, P< 0.05; Fig. 2). Similarly, plants inoculated with strain E19T combined with lanthanum 51 | S e i t e OD600 showed increases for leaves biomass by 17, 18, 29% and roots biomass by 48, 46, 58% compared to DB, DB+La and control. Plants grown in presence of only lanthanum showed also a good performance with increased leaves and root dry biomass (Tukey HSD, P< 0.05; Fig. 2) by 7, 7, 20% and 49 ,48, 60% compared to DB, DB+La, and control respectively. Plants inoculated with dead biomass of H. diazotrophicus with and without lanthanum had no significant effect in plant growth under salt stress compared to non-inoculated plants. Fig 2. Fresh and dry weight of leaves and roots (mean ± SD, n = 5) of barley plants with inoculation of H. diazotrophicus (E19T), in combination with La3+ (E19T + La), dead biomass of E19T (DB) and mixed with La3+ (DB + La3+), single La3+ (La) and control without amendments. Each value is the average of five replicates. Bars with the same letter are not significantly different (Tukey HSD, P< 0.05) among treatments after ANOVA. 52 | S e i t e Quantification of H. diazotrophicus on roots and rhizosphere of barley Survival and growth of H. diazotrophicus on roots and rhizosphere of barley plants 35 days after seed inoculation were determined using a specific qPCR assay. Specificity was confirmed in silico with the online tool TestPrime from Silva (Klindworth et al., 2013) and in situ by cloning environmental 16S rRNA genes and subsequent sequencing of the plasmid insert which recovered the expected sequence (Data not shown). A clear difference was observed between E19T colonization in rhizosphere (F5,20=5.33, P=0.003) and roots (F5,20=6.78, P=0.001) with much higher DNA target numbers of the root (Fig. 3). In rhizosphere quantification of H. diazotrophicus gene copies were twice in treatment with H. diazotrophicus in comparison to the bacterial treatment with lanthanum (E19T+La); whereas results for H. diazotrophicus gene copies in/on roots (Tukey HSD, P<0.05, Fig. 4 A, B) were similar on both treatments (E19T) and (E19T+La). Furthermore, the new primer for detection and quantification of E19T xoxF-gene (encoding the La-dependent methanol dehydrogenase) was tested, its specificity was evaluated in silico by PrimerBlast from NCBI and in situ as described for strain E19T 16S rRNA gene. The xoxF-gene involved in methylotrophic activity was detected on root samples with gene copies numbers of the xoxF-gene in a similar tendency (F5,20=3,18, P=0.024) to that observed for strain E19 T 16S gene in/on roots (Fig. S2). Fig 3. Quantification of strain E19T in rhizosphere soils (A) and roots (B) of barley 35 days after seed inoculation and plant growth. A specific primer for strain E19T targeting the 16S rRNA gene was used for qPCR. Each value is the average (± SD) of five replicates measured quadruple. Bars with the same letter are not significantly different (Tukey HSD; P<0.05) among treatments after ANOVA. E19 refers to samples with addition of H. diazotrophicus E19T, E19+La to samples with addition of H. diazotrophicus and lanthanum, DB to sample with addition of dead biomass, DB+La to sample with addition of dead biomass and lanthanum, La to sample with addition of lanthanum, Control to samples without addition. 53 | S e i t e Effect of seed inoculation with H. diazotrophicus on rhizosphere and root microbial community Total rhizosphere and root microbial community consisted of 45 phyla, 98 classes, 255 orders, 451 families and 845 genera. At phyla level most dominant in roots and rhizosphere were members of Proteobacteria with a relative abundance of 62%. In roots other dominant phyla were Actinobacteria (20.5%), Acidobacteria (6.9%), Verrucomicrobia (4.0%), Bacteroidetes (3.6%) and others less abundant phyla (5.24%) comprising the phyla Gemmatimonadetes, “Candidatus Patescibacteria”, Chloroflexi, Firmicutes, Lentisphaerae, “Candidatus Dependentiae”, Armatimonadetes, Cyanobacteria among others. In rhizosphere community besides Proteobacteria also Acidobacteria (14.6%), Verrucomicrobia (5.6%), Actinobacteria (4%), Bacteroidetes (3.3%) and others (6.2%) were found. The phylum Tenericutes was found only in rhizosphere. At classes level Gammaproteobacteria and Alphaproteobacteria were most abundant in roots with 40.6% and 18.3% and rhizosphere with 43.4% and 21.9% respectively (Fig. S3). At the order level, Xanthomonadales was the most abundant either in rhizosphere or roots with 35.7%. Family-level analysis revealed that with a mean relative abundance of 29.3% the family Rhodanobacteraceae was the most dominant in both habitats (rhizosphere and roots), other families such as Nocardioidaceae, Xanthomonadaceae, Caulobacteraceae, Opitutaceae, Xanthobacteraceae, Acidobacteriaceae (Subgroup.1), an uncultured family belonging to the order Vicinamibacterales, Solimonadaceae and Acetobacteraceae also were founded dominant in roots, whereas in rhizosphere; Acidobacteriaceae (Subgroup.1), Caulobacteraceae, an uncultured family belonging to the order Vicinamibacterales, Xanthobacteraceae, Opitutaceae, Xanthomonadaceae, Acetobacteraceae, Nocardioidaceae and Gemmatimonadaceae were the most dominant families (Fig. 4). 54 | S e i t e Fig 4. Phylogenetic distribution at family level of bacterial community from rhizosphere (A) and roots (B). E19 refers to samples with addition of H. diazotrophicus E19T, E19T + La to samples with addition of H. diazotrophicus and lanthanum, DB to sample with addition of dead biomass, DB + La to sample with addition of dead biomass and lanthanum, La to sample with addition of lanthanum, Control to samples without addition. Only the ten most abundant classes are shown, while the further 220 (Rhizosphere) and 159 (roots) families were collapsed into “Others”. Alpha and Beta-diversity The alpha diversity of the bacterial community was evaluated using several diversity indicators. Bacterial taxonomic diversity on rhizosphere and roots showed significant changes among the treatments in alpha-diversity indices observed ASVs (F5,16=6.45, P=0.002) (F5,23=3.47, P=0.0017) and Shannon only in rhizosphere (F5,16=4.99, P=0.006) (F5,23=1.76, P=0.161). In rhizosphere most significant differences of alpha diversity were found for the treatments with E19T and E19T with lanthanum (Tukey HSD P<0.05; Fig. 5). Roots showed a similar trend (Fig. 5) where the most significant differences in alpha diversity were found for E19T, dead biomass and E19T with lanthanum. The inoculation of barley seeds with E19T evidently showed changes of alpha diversity metrics for bacterial root and rhizosphere community in comparison to control seeds without inoculation. 55 | S e i t e Fig 5. Alpha diversity indices of the different treatments based on ASVs of bacterial community from rhizospheric soil and roots. Boxes with the same letter are not significantly different (Tukey HSD P<0.05) among treatments after ANOVA. E19 refers to samples with addition of H. diazotrophicus E19T, E19 + La to samples with addition of H. diazotrophicus and lanthanum, DB to sample with addition of dead biomass, DB + La to sample with addition of dead biomass and lanthanum, La to sample with addition of lanthanum, Control to samples without addition. According to the Bray-Curtis dissimilarities, nonmetric multidimensional scaling (NMDS) was employed to depict the beta diversity of the bacterial community (Fig. 6). The variation of communities in rhizosphere (Adonis, F5,16=3.82, P<0.001)) and roots (Adonis F5,22=2.93, P<0.001) of barley plants was significant between the different treatments. In both rhizosphere and roots similar bacteria communities were found in treatments with E19T and E19T with lanthanum; most different was the community in control soil. Other cluster correspond to the treatments of dead biomass and death biomass with lanthanum. 56 | S e i t e A. B. Fig 6. Non-Metric multidimensional scaling (NMDS) plot of β-diversity by Bray-Curtis dissimilarities for barley microbiota in (A) Rhizosphere (Adonis (P<0.001)) and (B) Roots (Adonis (P<0.001)). The number of permutations was 999. E19 T refers to samples with addition of H. diazotrophicus E19T, E19 T + La to samples with addition of H. diazotrophicus and lanthanum, DB to sample with addition of dead biomass, DB + La to sample with addition of dead biomass and lanthanum, La to sample with addition of lanthanum, Control to samples without addition. Differential Abundance Ranking Differential abundance analysis was tested on the genus level to identify the most associated genus by the different treatments using the plugin Songbird (Morton et al., 2019) for QIIME2 computing relative differentials ranks. The genus with higher differential ranking associated to the different treatments in rhizosphere and roots are showed in Table 1, and the top ten of genus are included in Table S5 and S6. As a whole Rhodanobacter was the most strongly associated genus between the treatments whereas Granulicella, Aquicella, uncultured bacteria of order Vicinamibacterales were associated to rhizosphere and Alkanibacter, uncultured bacteria of order Vicinamibacterales to roots respectively (Tables S5 and S6) . Table 1. Top-ranked genus abundance per treatment obtained from a multinomial regression rank test. Strain E19T refers to samples with addition of H. diazotrophicus E19T, E19T + La to samples with addition of H. diazotrophicus and lanthanum, DB to sample with addition of dead biomass, DB + La to sample with addition of dead biomass and lanthanum, La to sample with addition of lanthanum, Control to samples without addition. 57 | S e i t e Genus Ranked Treatment Rhizosphere Roots E19T Rhodanobacter sp. Tistrella sp. E19T+La Alkanibacter sp. Dongia sp. DB Rhodanobacter sp. Rhodanobacter sp. DB+La Caulobacter sp. Rhodanobacter sp. La Solirubrobacter sp. Family Blastocatellaceae-Uncultured bacteria Control Rhodanobacter sp. Nocardioides sp. DISCUSSION Rare earth elements (REE) were long time overseen as trace metals for enzyme activity of microorganisms. Since the publications of Hibi et al. (2011), Fitriyanto et al. (2011) and Pol et al. (2014) the importance of the REE for the lanthanides depending methanol metabolisms in bacteria in different environments (Howat et al., 2018; J. Huang et al., 2019) become evident. Therefore, we postulate that H. diazotrophicus as plant growth promoting bacteria and able to grow with methanol expressing a La- dependent methanol dehydrogenase is supported/favored in root colonization in the presence of La. The results obtained in this work support some of the hypotheses stated. Barley seed inoculation with strain E19T in presence of La enhanced plant biomass more that the treatment with E19T but without La (hypothesis I). Fresh and dry weight of both leaves and roots grown under salt stress were highest after E19T inculcation and presence of La. However the difference of leave and root biomass in the presence of strain E19T supplemented with La were not significant different compare to E19T, thus this hypothesis was not supported by our data. The untreated control plants and plants after seed inoculation of dead biomass of strain E19T were significantly lower compare to treatments with alive strain E19T and/or lanthanum. The observed growth stimulation of barley plants inoculated with strain E19T confirmed results of Suarez et al. (2015) who also reported significantly increased dry biomass of roots and leaves of barley plants after inoculation with strain E19T under salt stress conditions. The second working hypothesis (presence of lanthanum has no effect on plant growth) was not fulfilled, interestingly, in the presence of lanthanum dry and fresh weights were increased compared to control treatment. Previous studies reported that lanthanum might play a role in regulation of the plant antioxidant defense system, increasing its activity removing reactive oxygen species like oxygen peroxide (H2O2) , superoxide (O2 •-), and therefore diminishing the salt stress (Huang and Shan, 2018). Huang and Shan (2018) demonstrated a positive effect in plant height, stem diameter and dry weight after application of LaCl3 to tomato seedlings under salt stress conditions. Other studies carried out by Xu et al. (2007) and Liu et al. (2016) indicated that lanthanum application alleviated the oxidative damage in maize leaves and promoted the growth of Salvia involucrata under salt stress protecting the photosynthetic system from damage. An additional effect could be that other plant growth promoting 58 | S e i t e bacteria were stimulated with lanthanum. Rare-earth element depending alcohol dehydrogenases seems to be widely distributed (Howat et al., 2018; Huang et al., 2019) and rare earth depending enzyme activity were also found for another substrate (glycerol) in Pseudomonas putida known as well by its activity as PGPR (Wehrmann et al., 2020). Plants inoculated with dead bacteria did not improve barley growth, which supports work of Suarez et al. (2015) who stated that salt stress tolerance of barley is due to PGPR activity of bacteria and not an effect of nutrients provided in the dead biomass. Treatments of barley seeds inoculated with dead biomass with and without the presence of lanthanum were the same and the effect of lanthanum supporting plant growth disappeared. This observation could be explained by microprecipitation of lanthanum with the dead bacterial biomass. A similar observation was described by Kazy et al. (2006) where inactive biomass of Pseudomonas sp. accumulated high amount of lanthanum in a mechanism of La-biosorption. Our third hypothesis that lanthanum improves strain E19T rhizosphere abundance was not supported since the abundance of E19T in rhizosphere was lower in presence of lanthanum compared to no lanthanum treatment (Fig. 3 A). We suggest that lanthanum did not improve growth of strain E19T in the rhizosphere likely due to a rare earth element (REE) switch; a regulatory mechanism which modulate the expression of methanol dehydrogenase (xoxF) (Yu and Chistoserdova, 2017) conditioning the primary substrate consumption in strain E19T to methanol uptake. In pure culture E19T showed La- dependent growth on methanol (Fig. 1). If methanol concentrations however would be very low in the rhizosphere, possibly also because of substrate competition of other methylotrophic bacteria growth of strain E19T might occur based on other low molecular weight compounds like glucose, arabinose, mannose which have been described as typical rhizosphere compounds (Derrien et al., 2004; Sasse et al., 2018). These substrates can be utilized also in absence of lanthanum. On roots strain E19T was able to grow successfully and high copies numbers of bacteria were determined compared to rhizosphere (roots 4.3 x 106 copy numbers vs. rhizosphere 3.2 x 104 copy numbers gdw-1). Treatment with lanthanum did not affect growth of strain E19T on roots, methanol production during root growth through pectin demethylation of wall cells as described by Fall and Benson, 1996 and Galbally and Kirstine (2002) might attract E19T. However also other root exudates enable growth of strain E19T independent of La. Similarly high copy numbers of E19T xoxF gene were quantified (roots 4.8 x 106 copy numbers g-1), the ecological role of xoxF in establishment of bacteria in/on plants roots has not been up to date elucidated, however studies of Methylobacterium groups on phyllosphere of Arabidopsis thaliana, showed a high expression of xoxF over mxaF suggesting xoxF gene can play an important role during plant colonization (Delmotte, 2009). Further studies are required to clarify how strain E19T can settle on roots and establish a synergistic interaction with the plant. Various strategies for roots colonization have been described for different PGPR by Compant et al. (2010) and Gamez et al. (2019). For Pseudomonas fluorescens the presence of an exudated mucigel of roots (Hansen et al., 1997) has been described for attraction. 59 | S e i t e Our fourth hypothesis was confirmed, and significant differences were found in alpha diversity indices and beta diversity metrics. Seed inoculation with strain E19T influenced bacterial alpha diversity possibly due to the ability of strain E19T to stimulate exudates release from roots which subsequently enriches the microbial community diversity. Similar studies have reported that inoculation of plants with PGPR modified the composition and/or function of the rhizosphere bacterial community. Bhattacharyya et al. (2018), founded that inoculation of cabbage with Proteus vulgaris induced changes in community abundance. Inoculation of maize plants with Azospirillum lipoferum shaped the composition of the indigenous rhizobacterial community changing exudate patterns that result in the variability of the bacterial community as demonstrated by Baudoin et al. (2009) in a field trial. In our experiment seed treatment with dead biomass of strain E19T also showed a higher alpha diversity metrics possibly because nutrients from inactivated bacterial biomass might have enriched the bacteria populations. For lanthanum treatments only in roots, observed ASVs index were significant different to control treatment, which might indicate a specific enrichment of methylotrophic microbiota inside the roots using methanol for their grown (Butterfield et al., 2016). Interestingly, the Observed ASVs and Shannon index are higher in rhizosphere than in roots, the differences in the habitats could imply more restrictions for the growth and proliferation of specific adapted bacteria recruited by the plant in the roots (Compant et al., 2010). Beta diversity showed that all the treatments evidenced changes in bacterial communities and the differences are linked to the effects described for alpha diversity analyses. The taxonomical composition at the phylum level indicated that the most abundant phyla founded were Proteobacteria, Actinobacteria, Firmicutes, Acidobacteria, and Bacteroidetes which have also been detected by Ma and Gong (2013) in a soil affected by salt. On the taxonomic order level Xanthomonadales represent the abundant order between the treatments which coincide with the study carried out by Valenzuela-Encinas et al. (2009) who described the bacterial composition in three saline soil samples and found bacterial members belonging to the order Xanthomonadales. At the family level the dominant family was Rhodanobacterace. A multinomial regression designed for differential abundance analysis was used to identify the most abundant bacterial genera associated to the treatments; from the top ten most abundant genus (Table. S5 and S6) we observed that Rhodanobacter was by far the most dominant associated to the treatments in both rhizosphere and roots, in rhizosphere were also predominant the genera Granulicella and Caulobacter, while for roots an uncultured bacterial sequence of order Vicinamibacterales, no genera was uniquely present in either the soil or root samples. The first Rhodanobacter isolate was described as new genera and species able to catalyze the first steps of lindane (ɣ-HCH) degradation and was isolated from soil (Nalin et al., 1999). Many other Rhodanobacter species have been isolated later from soil including rhizosphere soil (Huo et al., 2018; Won et al., 2015) and the rhizoplane (Madhaiyan et al., 2014). DNA hydrolyzing activity was described for Rhodanobacter hydrolyticus (Dahal et al., 2018) which might contribute to abundance under treatments with dead biomass of E19T. Granulicella is the largest group from family Acidobacteriaceae, most of Granulicella species were isolated from peat bogs and tundra soils (Yamada et al., 2014); some species can tolerate high NaCl concentrations being able to growth at concentrations of 3.5% (W/V) 60 | S e i t e (Pankratov and Dedysh, 2010) what allowed them the develop in the saline conditions settled for our study. Species of genus Caulobacter have a particular mode of reproduction where the division generate one non motile prostheca cell and one cell with a polar flagellum (Staley, 1968), and can be found in environments, such as soil, water, rhizosphere soil, roots (Gao et al., 2018; Jin et al., 2013; Sun et al., 2017; Yang et al., 2020). A study reported by Benidire et al. (2020) of microbiota structure of Vicia faba after inoculation with PGPR in the presence or absence of saline stress showed Caulobacter as one of the main groups in root colonization in absence of saline stress, which coincide with not dominance of Caulobacter in roots in our study across the treatments, moreover the plant growth promotion activity have been also examined, a novel specie of Caulobacter isolated from maize roots has demonstrated to promote lateral root formation in the root, and increased size and number of shoots in plants of Arabidopsis thaliana as described by Luo et al. (2019). The order Vicinamibacterales and family Vicinamibacteraceae which include two species Vicinamibacter silvestris and Luteitalea pratensis, members of this order have been described as gram- negative, non-spore-forming and chemoheterotrophic (Dedysh and Yilmaz 2018); sequences of bacteria belonging to order Vicinamibacterales also were retrieved in a study of microbial communities of a soil cultivated with napier grass and amended with biochar (Yu et al., 2020). In conclusion, this study is the first study carried out to understand the interaction between a plant and a xoxF-type methylotrophic bacterium with PGPR activity in presence of a rare metal like lanthanum and corroborated that seed inoculation with H. diazotrophicus E19T promoted the growth on barley plants under saline conditions. Novel results include the (i) bacterial diversity in rhizosphere and roots of barley plants were higher in plants after seed inoculation with strain E19T, and (ii) lanthanum treatment had no incidence improving PGPR activity of strain E19T. Further studies are required to elucidate the action mechanism of strain E19T on growth of barley. Acknowledgements We are very grateful to Rita Geisser-Plaum, Renate Baumann, and Bellinda Schneider for the valuable technical support. For the helping with the lanthanum measurements we are thankful to Romy Auerbach. The Universidad Nacional de Colombia- Sede Medellín is thanked for the participation in the project. Funding The scholarship program “Pasaporte a la ciencia” from Colombian government provided financial support of Julian Rojas Conflict of interest The authors declare that they have no conflict of interest. Bibliography Ashraf, M. (1994). Breeding for Salinity Tolerance in Plants. 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Soil Biology and Biochemistry, 45, 139–146. https://doi.org/10.1016/j.soilbio.2011.11.006 67 | S e i t e Biology and Fertility of Soils- Supplementary Data Effect of Hartmannibacter diazotrophicus and lanthanum on the plant growth and microbial communities of barley grown under salt stress Julian Rojas1; Christian Suarez1; Stefan Ratering1; Marina Krutych1; Angelina Hormaza2; Sylvia Schnell1* 1Institute of Applied Microbiology, IFZ, Justus-Liebig University Giessen, 35392 Giessen, Germany 2 Faculty of Science, Universidad Nacional de Colombia - Sede Medellín, 050034 Medellín, Colombia Table S1. Physico-chemical properties of the substrate Fruhstorfer Erde Hawita typ P (HAWITA Gruppe GmbH, Vechta, Germany), the substrate used for the plant assays. Physico-chemical parameter Value pH (CaCl2) 5.9 KCl 1.0 g. l-1 Nitrogen (CaCl -12) 120 mg l Phosphate (CAL) 120 mg l-1 Potassium (CAL) 120 mg l-1 Magnesium (CaCl2) 120 mg l-1 Table S2. Sorption test of Lanthanum by ICP-MS analysis on non-sterile soil in triplicate replications (Fruhstorfer Erde Hawita typ P) Sample La (isotop 139) mg l-1 Stock 20 ppm 20.09 Stock 2 ppm 2.81 Typ P 2 ppm 0.81 Typ P 2 ppm 0.22 Typ P 2 ppm 0.28 Typ P 20 ppm 4.43 Typ P 20 ppm 4.08 Typ P 20 ppm 4.71 68 | S e i t e Figure S1. Growth of H. diazotrophicus E19T in freshwater media with La3+ (lanthanum 30 µM) and MeOH (methanol 5%v/v). Values represent the average of triplicate replications with corresponding standard deviations, the optical density (OD600) was used as growth parameter Table S3. Data sets evaluated by normality Anderson-Darling test prior to ANOVA analysis. Data sets with P-values less than alpha 0.05 (P<0.05) not come from a normal distribution. Data Anderson Darling-Value P-value Fresh Weigh Shoots 0.201 0.869 Fresh Weigh Roots 0.201 0.869 Dry Weigh Shoots 0.669 0.073 Dry Weigh Roots 0,504 0.189 qPCR-Copies/g Rhizosphere 0.213 0.757 qPCR Copies/g Roots 0.656 0.065 Alpha diversity Observed ASVs-Rhizosphere 0.567 0.125 Alpha diversity Shannon-Rhizosphere 0.809 0.325 Alpha diversity Observed ASVs-Roots 0.586 0.117 69 | S e i t e Table S4. Fresh and dry weight values of leaves and roots of barley plants with inoculation of H. diazotrophicus (E19T), supplemented with La3+ (E19T + La), dead biomass of E19T (DB) and mixed with La3+ (DB + La3+), single La3+ (La) and control without amendments. Average ± standard error from 5 replications. Growth E19T E19T + La DB DB + La La Control Parameter Leaves fresh weight (g-1) 12.35 ± 2.70 13.07 ± 2.98 12.06 ± 3.15 12.02 ± 3.16 12.48 ± 2.74 11.61 ± 3.12 Leaves dry weight (g-1) 1.84 ± 0.50 1.98 ± 0.52 1.79 ± 0.51 1.79 ± 0.50 1.86 ± 0.48 1.62 ± 0.46 Roots fresh weight (g-1) 5.72 ± 2.70 5.89 ± 2.98 4.38 ± 3.15 4.33 ± 3.16 5.76 ± 2.74 3.94 ± 3.12 Roots dry weight (g-1) 0.63 ± 0.50 0.69 ± 0.52 0.54 ± 0.51 0.56 ± 0.50 0.73 ± 0.48 0.52 ± 0.46 Figure S2. Quantification xoxF-gene of strain E19T in roots of barley 35 days after seed inoculation and plant growth. Each value is the average (± SD) of five replicates measured quadruple. Bars with the same letter are not significantly different (Tukey HSD; P<0.05) among treatments after ANOVA. E19 refers to samples with addition of H. diazotrophicus E19T, E19+La to samples with addition of H. diazotrophicus and lanthanum, DB to sample with addition of dead biomass, DB+La to sample with addition of dead biomass and lanthanum, La to sample with addition of lanthanum, Control to samples without addition. 70 | S e i t e 100% Gammaproteobacteria Alphaproteobacteria Acidobacteria Acidobacteria; Subgroup 6 50% Verrucomicrobiae Actinobacteria Bacteroidia 0% Gemmatimonadetes Deltaproteobacteria Others A. 100% Gammaproteobacteria Alphaproteobacteria Actinobacteria 50% Verrucomicrobiae Acidobacteria Acidobacteria; Subgroup 6 Bacteroidia 0% Gemmatimonadetes Parcubacteria Others B. Figure S3. Phylogenetic distribution at class level of bacterial community from rhizosphere (A) and roots (B). Only the ten most abundant classes are shown, while the further 44 (Rhizosphere) and 35 (roots) classes were collapsed into “Others”. 71 | S e i t e % Relative abundance % Relative abundance E19 E19 E19 E19 E19 E19 E19 E19+La E19 E19+La E19+La E19 E19+La E19+La E19+La E19+La DB DB E19+La DB E19+La DB DB DB DB DB+La DB+La DB DB+La DB DB+La DB+La DB+La La DB+La La DB+La La La La Control La Control Control Control Control Control Control Control Control Table. S5 Top ten ranked taxa genus per treatment in rhizosphere. Treatment E19 Taxonomy Feature ID 722f3de083fe8bcbfbd08cb6e2f5f0fe Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Rhodanobacter cfd3c6d979b698047f6b5d54290932a0 Acidobacteriota; Vicinamibacteria; Vicinamibacterales; uncultured; uncultured 9cfe32c6dad0f09b722dc51ac417823c Proteobacteria; Gammaproteobacteria; Legionellales; Coxiellaceae; Aquicella 82996ae173075afd94a55ba684a5ea59 Verrucomicrobiota; Verrucomicrobiae; Opitutales; Opitutaceae; Opitutus e641e131483e297b5e69ef6fcb901bea Acidobacteriota; Acidobacteriae; Acidobacteriales; Acidobacteriaceae_(Subgroup_1); Granulicella 6aa42a3bb44fb31e743bb20bddeafea4 Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Caulobacter 5c502159d8025e710f37f509c20021e7 Acidobacteriota; Acidobacteriae; Solibacterales; Solibacteraceae; Candidatus_Solibacter abee41022a9cce3620183c485f494d2d Proteobacteria; Alphaproteobacteria; Acetobacterales; Acetobacteraceae; Rhodovastum 288b3b0cb1cae1e302ceafc49de2f2a9 Proteobacteria; Alphaproteobacteria; Dongiales; Dongiaceae; Dongia 4 557e11e97b0be54e2890bbc1ab17aa4 Proteobacteria; Alphaproteobacteria; Rhizobiales; Xanthobacteraceae; Bradyrhizobium Treatment E19+La d8fe8fce20f5033355c8b591dc70fa78 Proteobacteria; Gammaproteobacteria; Salinisphaerales; Solimonadaceae; Alkanibacter e641e131483e297b5e69ef6fcb901bea Acidobacteriota; Acidobacteriae; Acidobacteriales; Acidobacteriaceae_(Subgroup_1); Granulicella 12ddf670a2a1a4c7b562b286ba5367ac Proteobacteria; Alphaproteobacteria; Reyranellales; Reyranellaceae; Reyranella 722f3de083fe8bcbfbd08cb6e2f5f0fe Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Rhodanobacter 8ebaa5061aa34b1fbfa1eec3ed47983b Acidobacteriota; Acidobacteriae; Solibacterales; Solibacteraceae; Candidatus_Solibacter 5493a905ede49d0f9141effc82aae995 Actinobacteriota; Actinobacteria; Micrococcales; Microbacteriaceae f8cf9ff7ffc137d8486a1a6dc047bb3f Dependentiae; Babeliae; Babeliales; Vermiphilaceae; Vermiphilaceae abbbbce464d2e6b809867604f6876b96 Patescibacteria; Parcubacteria; Candidatus_Kaiserbacteria; Candidatus_Kaiserbacteria; Candidatus_Kaiserbacteria dd7beb41e3d66aa46d69907586418e21 Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Caulobacter a5d3593d869d7e1e9633b970b449b9bc A ctinobacteriota; Actinobacteria; Micrococcales; Promicromonosporaceae; Promicromonospora Treatment Death biomass 220b5a09598e39a9f564e1e86007a46c Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae 4cc8857431d97ba771ed1d941b15875d Acidobacteriota; Vicinamibacteria; Vicinamibacterales; uncultured; uncultured e8594d94fae64ee074836846f8efa5df Actinobacteriota; Actinobacteria; Streptomycetales; Streptomycetaceae; Streptomyces 2ee5b26882e6c8a61f3a984af852280a Acidobacteriota; Acidobacteriae; Acidobacteriales; Acidobacteriaceae_(Subgroup_1); Granulicella d8fe8fce20f5033355c8b591dc70fa78 Proteobacteria; Gammaproteobacteria; Salinisphaerales; Solimonadaceae; Alkanibacter e3d8616809b19796ee96b370f8af563c Verrucomicrobiota; Verrucomicrobiae; Opitutales; Opitutaceae; Lacunisphaera eb4541bfc1922a99b0bc7ef1e6c06d79 Sumerlaeota; Sumerlaeia; Sumerlaeales; Sumerlaeaceae; Sumerlaea 179f89ab409c974dbe519f00812e5434 Bdellovibrionota; Oligoflexia; 0319-6G20; 0319-6G20; 0319-6G20 f1e170d5f3c8736838136a76df08d8b3 Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Luteimonas a5d3593d869d7e1e9633b970b449b9bc Actinobacteriota; Actinobacteria; Micrococcales; Promicromonosporaceae; Promicromonospora Treatment .DB+La dd7beb41e3d66aa46d69907586418e21 Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Caulobacter 9cfe32c6dad0f09b722dc51ac417823c Proteobacteria; Gammaproteobacteria; Legionellales; Coxiellaceae; g_Aquicella 4cc8857431d97ba771ed1d941b15875d Acidobacteriota; Vicinamibacteria; Vicinamibacterales; uncultured; uncultured 8c6b0c63ca6922b21c76c66539a60923 Proteobacteria; Alphaproteobacteria; Rhizobiales; Xanthobacteraceae; Rhodopseudomonas fb82af326f24c8e2492d613ac2e15271 Verrucomicrobiota; Verrucomicrobiae; Opitutales; Opitutaceae e8594d94fae64ee074836846f8efa5df Actinobacteriota; Actinobacteria; Streptomycetales; Streptomycetaceae; Streptomyces 2ee5b26882e6c8a61f3a984af852280a Acidobacteriota; Acidobacteriae; Acidobacteriales; Acidobacteriaceae_(Subgroup_1); Granulicella 85c64dfcc7e38e56239cbc7d661d6816 Actinobacteriota; Actinobacteria; Propionibacteriales; Nocardioidaceae; Kribbella 179f89ab409c974dbe519f00812e5434 Bdellovibrionota; Oligoflexia; 0319-6G20; 0319-6G20; 0319-6G20 d9c290dad797d0636e94fa8c5cdb0c49 P roteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Phenylobacterium Treatment La 28502a411927dadb7af7eea937c8a905 Actinobacteriota; Thermoleophilia; Solirubrobacterales; Solirubrobacteraceae; Solirubrobacter c7f519f50a9112eed281131d9329ca32 Proteobacteria; Alphaproteobacteria; Rhizobiales; Devosiaceae; Devosia fe9f0ebd7aab56ce96f498bced86bf80 Verrucomicrobiota; Verrucomicrobiae; Opitutales; Opitutaceae 9e851dfbc37ff8cc2a47cd21e1cc3fe7 Proteobacteria; Gammaproteobacteria; Legionellales; Coxiellaceae; g_Aquicella 80201efe5481555cee510e108c297090 Proteobacteria; Alphaproteobacteria; Rhodospirillales; Rhodospirillaceae; uncultured 40f27c637cde80714a2313cf67d5f692 Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Phenylobacterium 9bc8bf2b9ad588a867f23ee077754929 Proteobacteria; Alphaproteobacteria; Acetobacterales; Acetobacteraceae; uncultured 4055f3ab461b2adf7c59a5dfa410e2e6 Acidobacteriota; Acidobacteriae; Acidobacteriales; Acidobacteriaceae_(Subgroup_1); Granulicella 5c502159d8025e710f37f509c20021e7 Acidobacteriota; Acidobacteriae; Solibacterales; Solibacteraceae; Candidatus_Solibacter dc03a075baf013d7de0df3debbb01070 Proteobacteria; Gammaproteobacteria; Burkholderiales; Burkholderiaceae; Burkholderia- C aballeronia-Paraburkholderia 72 | S e i t e Control 40ae6e0f80204fddef2b030fb08dd3ae Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Rhodanobacter 8d1be44b544f7271330c4a68697346c0 Verrucomicrobiota; Verrucomicrobiae; Opitutales; Opitutaceae; Opitutus ed583e3562a89139a870af286de44084 Acidobacteriota; Acidobacteriae; Acidobacteriales; Acidobacteriaceae_(Subgroup_1); Granulicella 96ac15fdf84ba15d752b4f098f053793 Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae 31af174831d38265000802fb9c54c974 Proteobacteria; Alphaproteobacteria; Rhizobiales; Xanthobacteraceae; Rhodopseudomonas 4557e11e97b0be54e2890bbc1ab17aa4 Proteobacteria; Alphaproteobacteria; Rhizobiales; Xanthobacteraceae; Bradyrhizobium f1e170d5f3c8736838136a76df08d8b3 Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Luteimonas 6eceb3ef8939eee83bb630c6e9f63769 Bacteroidota; Bacteroidia; Chitinophagales; Chitinophagaceae; Chitinophaga 90e2666427144e93bed1cd32da1f2c39 Actinobacteriota; Actinobacteria; Propionibacteriales; Nocardioidaceae; Kribbella 2167e8f2ceb12316eff450b006e71ed3 Gemmatimonadota; Gemmatimonadetes; Gemmatimonadales; Gemmatimonadaceae; uncultured Table. S6 Top ten ranked taxa genus per treatment in roots. Treatment E19 Taxonomy Feature ID ca9958262efc0ea520e109a45bfa6281 Verrucomicrobiota; Verrucomicrobiae; Pedosphaerales; Pedosphaeraceae; Pedosphaeraceae 27a614bf842ae59c6056672d720701fd Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Rhodanobacter 76a6e03ae98d8836ca380dacd294332d Bacteroidota; Bacteroidia; Sphingobacteriales; Sphingobacteriaceae; Pedobacter 3cfc18fb09ff6ec6c12d237afd940df5 Acidobacteriota; Vicinamibacteria; Vicinamibacterales; uncultured; uncultured 209f831103775cb6da3afb10f447fcb6 Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Brevundimonas a008a56100a38856379e0285c7221aaf Proteobacteria; Alphaproteobacteria; Rhizobiales; Xanthobacteraceae; Rhodopseudomonas ee0036283ef96e94d4b13784cbe2b5c1 Actinobacteriota; Acidimicrobiia; Microtrichales; Iamiaceae; Iamia 5689250dae3b42d639e404ae649fc0dd Actinobacteriota; Actinobacteria; Propionibacteriales; Nocardioidaceae; Nocardioides b00c77a07594dfa8e25854514ccb1888 Abditibacteriota; Abditibacteria; Abditibacteriales; Abditibacteriaceae; Abditibacterium ec41952da0cf83f457a4a4080d8b5e1f Patescibacteria; Parcubacteria; Candidatus_Kaiserbacteria; Candidatus_Kaiserbacteria; Candidatus_Kaiserbacteria Treatment E19+La 8cd5233fa58de785d0747e27b1473d12 Proteobacteria; Alphaproteobacteria; Dongiales; Dongiaceae; Dongia 5cefece331e3d797138525d689dcbeba Proteobacteria; Alphaproteobacteria; Acetobacterales; Acetobacteraceae; uncultured d2888eeaf0a056ccdb69c75feef9d049 Chloroflexi; Dehalococcoidia; S085; S085; S085 1dd1f1bc267a302964ae649ab647649c Planctomycetota; Planctomycetes; Planctomycetales; Schlesneriaceae; Schlesneria 165eb72f4ed1a5f9d1bbd90d49333bfd Gemmatimonadota; Gemmatimonadetes; Gemmatimonadales; Gemmatimonadaceae; uncultured 3694db554c4bbc01f2f1134822d19079 Firmicutes; Sulfobacillia; Sulfobacillales; Sulfobacillaceae; Sulfobacillus a65f5796ad14dba33e77b80686a206e6 Proteobacteria; Alphaproteobacteria; uncultured; uncultured; uncultured a6f6fa82a84b34cc25f48c8450e7b249 Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Rhodanobacter 88e745080e90b2f90e54a8b257f94b64 Sumerlaeota; Sumerlaeia; Sumerlaeales; Sumerlaeaceae; Sumerlaea 8e31565fcaaa5627529016e16e5af346 Actinobacteriota; Acidimicrobiia; Microtrichales; Iamiaceae; Iamia Treatment Death Biomass 27a614bf842ae59c6056672d720701fd Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Rhodanobacter 3cfc18fb09ff6ec6c12d237afd940df5 Acidobacteriota; Vicinamibacteria; Vicinamibacterales; uncultured; uncultured 90747717ff3ade88dea933c904d0f6f2 Proteobacteria; Gammaproteobacteria; Salinisphaerales; Solimonadaceae; Alkanibacter 02ca4e3cd35a5ac9e758ffe99c2ebdf5 Gemmatimonadota; Gemmatimonadetes; Gemmatimonadales; Gemmatimonadaceae; uncultured 5aee2ce96031a900dc66fb28cc5897d6 Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Luteimonas ddbe1f93ec71e195ea91b2ecfdd21e23 Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Pseudoxanthomonas 76a6e03ae98d8836ca380dacd294332d Bacteroidota; Bacteroidia; Sphingobacteriales; Sphingobacteriaceae; Pedobacter 2a8ab5c1bf2d8b56026d183ac75c0f9e Acidobacteriota; Acidobacteriae; Acidobacteriales; Acidobacteriaceae_(Subgroup_1); Granulicella c1abf51c267237256993ac5a591a4aca Actinobacteriota; Actinobacteria; Propionibacteriales; Nocardioidaceae; Aeromicrobium 35f62206ebe4a0845822a88e230d78ed P roteobacteria; Alphaproteobacteria; Rhodospirillales; uncultured; uncultured Treatment DB + La 73 | S e i t e 27a614bf842ae59c6056672d720701fd Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Rhodanobacter 354d85911f43d9fbc72d1a58337b883f Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Dyella 9af5c02f01fbca4fc803869f3bd25b90 Proteobacteria; Gammaproteobacteria; Burkholderiales; Nitrosomonadaceae; Nitrosospira 0b37066af79eac2c068a0ddf79cf0fbf Proteobacteria; Gammaproteobacteria; Salinisphaerales; Solimonadaceae; Alkanibacter 3cfc18fb09ff6ec6c12d237afd940df5 Acidobacteriota; Vicinamibacteria; Vicinamibacterales; uncultured; uncultured c4fd969d3b59f48f5ff85ec5487946cc Proteobacteria; Alphaproteobacteria; Reyranellales; Reyranellaceae; Reyranella eb912b6a8cdf34585299f27b3ea573c3 Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Phenylobacterium 79e6fecfd64ce85be66774789481edb7 Acidobacteriota; Acidobacteriae; Acidobacteriales; Acidobacteriaceae_(Subgroup_1); Granulicella 262f9d9c05883a19d56776389266069b Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Luteimonas 3ac0e153f0a77e63b1e7ab931dd73a68 P roteobacteria; Gammaproteobacteria; Legionellales; Legionellaceae; Legionella Treatment La b0f3c9c756d25481280498c7c682b085 Acidobacteriota; Blastocatellia; Blastocatellales; Blastocatellaceae; uncultured bef0ea6a4379edb4874c8fcd55d1aace Proteobacteria; Gammaproteobacteria; Burkholderiales; Alcaligenaceae 8363af5c48b88b9d7e127bd9b3b0ee17 Verrucomicrobiota; Verrucomicrobiae; Opitutales; Opitutaceae; Cephaloticoccus; 7bc7e6881183ae9794629f048cf63ce5 Acidobacteriota; Vicinamibacteria; Vicinamibacterales; uncultured; uncultured 958fc4d7612c3b84c8853f215d2ca2f9 Bacteroidota; Bacteroidia; Sphingobacteriales; Sphingobacteriaceae; Pedobacter a008a56100a38856379e0285c7221aaf Proteobacteria; Alphaproteobacteria; Rhizobiales; Xanthobacteraceae; Rhodopseudomonas ca9958262efc0ea520e109a45bfa6281 Verrucomicrobiota; Verrucomicrobiae; Pedosphaerales; Pedosphaeraceae; Pedosphaeraceae cd8a58fe596daa1c15855e36de32aae8 Proteobacteria; Alphaproteobacteria; Rhizobiales; Xanthobacteraceae; Nitrobacter ce3463c6cab44347c61b5fa79a48bb65 Bacteroidota; Bacteroidia; Cytophagales; Microscillaceae; uncultured ee0036283ef96e94d4b13784cbe2b5c1 Actinobacteriota; Acidimicrobiia; Microtrichales; Iamiaceae; Iamia Control e1e80cb02e4f8e3997cd124d3fcdc900 Actinobacteriota; Actinobacteria; Propionibacteriales; Nocardioidaceae; Nocardioides c78b590810def61034e0e1653e3400e6 Proteobacteria; Gammaproteobacteria; Xanthomonadales; Rhodanobacteraceae; Rhodanobacter 01dc497fa9db2a4238044630a9598628 Verrucomicrobiota; Verrucomicrobiae; Opitutales; Opitutaceae; Opitutus 5aee2ce96031a900dc66fb28cc5897d6 Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Luteimonas 209f831103775cb6da3afb10f447fcb6 Proteobacteria; Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Brevundimonas 9b5facd4baa2f80a0cb44762b134de10 Actinobacteriota; Actinobacteria; Propionibacteriales; Nocardioidaceae; Kribbella a32cb9f206aac5984238431d2e61ffe6 Sumerlaeota; Sumerlaeia; Sumerlaeales; Sumerlaeaceae; Sumerlaea c1abf51c267237256993ac5a591a4aca Actinobacteriota; Actinobacteria; Propionibacteriales; Nocardioidaceae; Aeromicrobium 7be716d1ee84aac44f591723e811542c Proteobacteria; Alphaproteobacteria; Rhizobiales; Xanthobacteraceae; Bradyrhizobium f37c3b9161b02a42696ae6c896df2909 Proteobacteria; Alphaproteobacteria; Reyranellales; Reyranellaceae; Reyranella 74 | S e i t e CHAPTER 3. Spirosoma endbachense sp. nov., isolated from a natural salt meadow Published in: International Journal of Systematic and Evolutionary Microbiology 75 | S e i t e 76 | S e i t e 77 | S e i t e 78 | S e i t e 79 | S e i t e 80 | S e i t e 81 | S e i t e 82 | S e i t e Supplement Material International Journal of Systematic and Evolutionary Microbiology Spirosoma endbachense sp. nov., isolated from a natural salt meadow Julian Rojas1, Binoy Ambika Manirajan2, Christian Suarez1, Stefan Ratering1, Rita Geissler- Plaum1 and Sylvia Schnell1 1 Institute of Applied Microbiology, Research Center for BioSystems, Land Use, and Nutrition (IFZ), Justus-Liebig- University Giessen, 35392, Germany 2School of Biosciences, Mahatma Gandhi University, Kerala, India Fig. S1. Maximum-parsimony phylogenetic tree based on 16S rRNA gene sequences, showing the phylogenetic position of strain I-24T among related members in the genus Spirosoma and other members of the family Cytophagaceae. Bootstraps values (based on 1000 replications) greater than 70% are showed at the branch points. Filled black points indicate that the corresponding nodes were also recovered in trees generated with maximum-likelihood and neighbor-joining algorithms. The tree was rooted using type strains of Sphingobacterium, Flammeovirga, Flexithrix and Adhaeribacter. Bar: 0.10 substitutions per nucleotide position. 83 | S e i t e Fig. S2. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences (a-termini filter between positions 67 and 1353, Escherichia coli numbering), showing the phylogenetic position of strain I-24T among related members in the genus Spirosoma and other members of the family Cytophagaceae. Bootstraps values (based on 1000 replications) greater than 70% are showed at the branch points. Filled black points indicate that the corresponding nodes were also recovered in trees generated with maximum-likelihood and maximum-parsimony algorithms. The tree was rooted using type strains of Sphingobacterium, Flammeovirga, Flexithrix and Adhaeribacter. Bar: 0.10 substitutions per nucleotide position. 84 | S e i t e Fig. S3. ANI (Average Nucleotide Identity) mean values matrix heatmap between strain I-24T, S. agri KCTC 52727T and S. terrae KCTC 52035T. 85 | S e i t e Fig. S4. Venn diagram depicted the number of coding sequences shared between I-24T, S. agri KCTC 52727T and S. terrae KCTC 52035T. 86 | S e i t e Fig. S5. Predicted gene clusters of secondary metabolites biosynthesis annotated in antiSMASH against strain I-24T draft genome. Analyses showed the identification of 4 clusters involved in biosynthesis of ladderane, terpene, polyketide synthase type I and III (T1PKS, T3PKS) and non-ribosomal peptide synthetase (NRPS) 87 | S e i t e A. B. C. Fig. S6. Polar lipids profile of strains A. I-24T B. S. agri KCTC 52727T and C. S. terrae KCTC 52035T separated by two-dimensional silica gel thin layer chromatography. L, unidentified lipid; AL, unidentified aminolipid; PL, unidentified phospholipid; GL, glycolipid; PE, phosphatidylethanolamine; PNL, phosphoaminolipid. Total lipid material and specific functional groups were revealed using spray reagents specific dodecamolybdophosphoric acid (total lipids), Zinzadze reagent (phosphate), ninhydrin (free amino groups), periodate-Schiff (α-glycols), Dragendorff reagent (quaternary nitrogen) and α – naphthol-sulphuric acid (glycolipids). Table S1. Polar lipids profile of strain I-24T compared to nearest Spirosoma species. L, unidentified lipid; AL, unidentified aminolipid; PL, unidentified phospholipid; GL, glycolipid; PE, phosphatidylethanolamine; PNL, phosphoaminolipid. *Data obtained in this study PE GL PL AL APL L Reference I-24T + + + + + + * Spirosoma agri + + + + + + * Spirosoma terrae + + + + + + * Spirosoma migulaei + - + - + + [1] Spirosoma pulveris + - + + + + [2] Spirosoma swuense + + + + + + [3] Spirosoma aerophilum + - - + + + [4] Spirosoma rigui + - - + - + [5] Spirosoma litoris + - + - + + [6] Spirosoma koreense + - + + + + [7] Spirosoma linguale + - - + - + [8] 88 | S e i t e Spirosoma - - + + + + [9] jeollabukense REFERENCES 1. Okiria J, Ten LN, Park S-J, Lee S-Y, Lee DH, et al. Spirosoma migulaei sp. nov., isolated from soil. J Microbiol 2017;55:927–932. 2. Joo ES, Lee J-J, Cha S, Jheong W, Seo T, et al. Spirosoma pulveris sp. nov., a bacterium isolated from a dust sample collected at Chungnam province, South Korea. J Microbiol 2015;53:750–755. 3. Joo ES, Kim EB, Jeon SH, Srinivasan S, Kim MK. Spirosoma swuense sp. nov., isolated from wet soil. Int J Syst Evol Microbiol 2017;67:532–536. 4. Hong S-B, Seok S-J, Kim J-S, Kwon S-W, Ahn J-H, et al. Spirosoma aerophilum sp. nov., isolated from an air sample. Int J Syst Evol Microbiol 2016;66:2342–2346. 5. Baik KS, Kim MS, Park SC, Lee DW, Lee SD, et al. Spirosoma rigui sp. nov., isolated from fresh water. Int J Syst Evol Microbiol 2007;57:2870–2873. 6. Okiria J, Ten LN, Lee J-J, Lee S-Y, Cho Y-J, et al. Spirosoma litoris sp. nov., a bacterium isolated from beach soil. Int J Syst Evol Microbiol 2017;67:4986–4991. 7. Ten LN, Okiria J, Lee J-J, Lee S-Y, Kang I-K, et al. Spirosoma koreense sp. nov., a species of the family Cytophagaceae isolated from beach soil. Int J Syst Evol Microbiol 2017;67:5198–5204. 8. Lail K, Sikorski J, Saunders E, Lapidus A, Glavina Del Rio T, et al. Complete genome sequence of Spirosoma linguale type strain (1T). Stand Genomic Sci 2010;2:176–184. 9. Li W, Ten LN, Lee SY, Lee DH, Jung HY. Spirosoma jeollabukense sp. nov., isolated from soil. Arch Microbiol 2018;200:431–438. 89 | S e i t e CHAPTER 4. Draft Genome Sequences of Spirosoma agri KCTC 52727 and Spirosoma terrae KCTC 52035 Published in: Microbiology Resource Announcements 90 | S e i t e 91 | S e i t e 92 | S e i t e CHAPTER 5. Screening of bacterial strains with methylotrophic activity lanthanum dependent 93 | S e i t e Summary The methanol is one of the most abundant volatile organic compounds in the atmosphere (Galbally and Kirstine, 2002) and it is also one of the carbon sources that methylotrophic bacteria oxidize to formaldehyde by means of methanol dehydrogenase (MDH) as their sole carbon and energy source (Kolb, 2009). In gram‐negative methylotrophic bacteria, MDH is well characterized, it possesses pyrroloquinoline quinone (PQQ) as a prosthetic group and is encoded by mxaF- gene. More recently a lanthanide-dependent methanol dehydrogenase, encoded by the gene xoxF has been discovered (Hibi et al., 2011a; Fitriyanto et al., 2011). No study has focused on bioprospection of methanol consuming microorganisms that may use the recently discovered lanthanum-dependent MDH-xoxF from saline and alkaline environments which is why this study aimed to isolate lanthanum- dependent methanol utilizing bacteria from saline habitats. Methodology Sample collection Soil samples were collected from natural salt meadows nearby Münzenberg, Hessen, Germany (50°27'46.2"N 8°45'55.1"E) and Bad Endbach, Hessen, Germany (50° 45’ 54.9’’ N 8° 26’ 41.2’’E), branches from a blackthorn wall at graduation buildings where salty spring water slowly trickles down (50° 21’ 48.8’’N 8°44’ 39.6’’E) as well a water sample from a fountain with salty spring water (50° 22’ 2.23’’N 8°44’ 36.8’’E) in Bad Nauheim, Hessen, Germany were collected. Isolation and identification of methanol utilizers The samples of soils and water were used as inoculum for enrichment cultures in modified liquid freshwater media (Widdel and Bak, 1992) containing 1.0 g NaCl, 0.4 g MgCl x 6H2O, 0.15 g CaCl2 x 2H2O, 0.5 g KCl, 0.2 g KH2PO4 , 0.25 g NH4 Cl, 1.4 g Na2SO4 , 1.0 ml trace element solution containing no lanthanum (SL10), 50 ml Phosphate buffer 0.4 M pH 7.0, 1.0 ml vitamin B12-solution, 1.0 ml 5-vitamin solution, 1.0 ml thiamine solution, 1.0 ml riboflavine solution in 1.0 liter deionized water, the pH was adjusted to 6.5; additionally 30 µM lanthanum and methanol 94 | S e i t e 5.0% V/V were added to the liquid medium. The enrichment cultures were shaken with 110 rpm and incubated at 28°C for one month. After several transfers in new medium the enriched bacteria were isolated on freshwater agar medium containing 30 µM lanthanum chloride and methanol 5% V/V. Different morphologies of colonies were obtained and various cell morphologies have been observed microscopically. For isolated strains the 16S RNA gene was amplified using the primer EUB 9f according to the method of Kampmann et al., 2012; PCR products were purified using the PCR purification kit (Qiagen) prior to sequencing by LGC genomics and the taxonomic affiliations were identified using EzBioCloud server (Yoon et al., 2017). Growth test with new isolates A test to evaluate the growth of the identified isolates on fresh water solid media with methanol in presence and absence of lanthanum was performed. For those isolates with good growth in presence of lanthanum on agar medium also liquid medium was tested and growth was evidenced by high turbidity and microscopic controls. Growth curves in liquid freshwater media with methanol 5% v/v and with/without addition of 30 μM LaCl3 were carried out. Optical density (OD) measurements were performed in a TECAN infinity 200 fluorescent spectrophotometer using a Greiner 48-well plate. Isolates were cultured for 5 days with orbital shaking amplitude 4 mm, eight OD measurements were taken at 600 nm (bandwidth 9) with 5 flashes at 25 °C. Results From collected samples, thirty-one strains were isolated, and their 16S RNA gene was sequenced for taxonomic affiliation. The next relatives of the isolated strains and the morphological and colony description are given in Table 1 and Table S1. Strain N° Source Blast Bacteria 16S % Similarity Next Relatives rRNA gene (EZ-Biocloud) 1 Salt meadow Bad Ancylobacter defluvii 99.33 Starkeya novella 99.07% Endbach Starkeya koreensis 98.53% 95 | S e i t e Ancylobacter rudongensis 98.4% 2 Salt meadow Bad Ancylobacter defluvii 99.47 Ancylobacter oerskovii 98.32% Endbach Ancylobacter rudongensis 98% Ancylobacter dichloromethanicus 97.9% 3 Salt meadow Bad Paenibacillus 99.72 Paenibacillus lautus 98.21% Endbach glucanolyticus Paenibacillus qingshengii 97.66% Paenibacillus lactis 96.96% 4 Salt meadow Bad Rhizobium nepotum 99.79 Rhizobium radiobacter 99.04% Endbach Rhizobium skierniewicense 98.72% Rhizobium rubi 98.29% 5 Salt meadow Bad Ancylobacter defluvii 99.47 Ancylobacter oerskovii 98.32% Endbach Ancylobacter rudongensis 98% Ancylobacter dichloromethanicus 97.9% 6 Salt meadow Variovorax 99.29 Variovorax paradoxus 99.15% Münzenberg boronicumulans Variovorax gossypii 99.01% Variovorax guangxiensis 99.01% 7 Salt meadow Paenibacillus validus 100 Paenibacillus xylanisolvens Münzenberg 97.38% Paenibacillus mucilaginosus 96.97% Paenibacillus filicis 96.97% 8 Graduation Ancylobacter 99.89 Ancylobacter dichloromethanicus Building rudongensis 99.47% Ancylobacter Bad Nauheim aquaticus 99.36% Ancylobacter vacuolatus 99.26% 9 Graduation Jiella aquimaris 98.67 Aurantimonas coralicida 96.45% Building Aurantimonas endophytica 96.45% Bad Nauheim Aureimonas glaciistagni 96.45% 10 Graduation Tistrella bauzanensis 99.33 Tistrella mobilis 97.33% building FM209132_s uncultured bacteria Bad Nauheim clon 89.97% Aliidongia dinghuensis 89.86% 11 Water Fountain Paracoccus homiensis 99.73 Paracoccus zeaxanthinifaciens Bad Nauheim 99.33% Paracoccus beibuensis 98.38% Pararhodobacter aggregans 98.12% 96 | S e i t e 12 Salt meadow Azospirillum oryzae 99.73 Azospirillum zeae 99.47% Münzenberg Azospirillum humicireducens 98.28% Azospirillum largimobile 98.25% 13 Salt meadow Pseudomonas 99.88 Pseudomonas plecoglossicida Münzenberg hunanensis 99.76% Pseudomonas monteilii 99.64% Pseudomonas taiwanensis 99.51% 14 Salt meadow Idonella dechloratans 99.25 Ideonella paludis 98% Münzenberg Ideonella azotifigens 97.99% Rubrivivax gelatinosus 97.75% 15 Graduation building Aurantimonas coralicida 99.05 Aurantimonas litoralis 99.05% Bad Nauheim Aurantimonas manganoxydans 98.78% Aureimonas frigidaquae 97.42% 16 Salt meadow Hyphomicrobium facile 99.58 Hyphomicrobium facile subsp. Bad Endbach subsp. facile Tolerans 99.58% Hyphomicrobium facile subsp. Ureaphilum 99.58% Hyphomicrobium methylovorum 98.31% 18 Salt meadow Variovorax 98.86 Variovorax paradoxus 98.74% Bad Endbach boronicumulans Variovorax gossypii 98.61% Variovorax ginsengisoli 98.61% 20 Salt meadow Hyphomicrobium faciles 99.58 Hyphomicrobium facile subsp. Bad Endbach subs. facile Tolerans 99.58% Hyphomicrobium facile subsp. Ureaphilum 99.58% Hyphomicrobium methylovorum 98.31% 21 Salt meadow Microbacterium 99,9 Microbacterium aerolatum 98.69% Bad Endbach natoriense Microbacterium ginsengiterrae 98.64% Microbacterium foliorum 98.54% 22 Salt meadow Variovorax 99.05 Variovorax paradoxus 98.74% Bad Endbach boronicumulans Variovorax gossypii 98.61% Variovorax ginsengisoli 98.61% 23 Salt meadow Nocardiodes simplex 98.60 Nocardioides caeni 98.6% Bad Endbach Nocardioides nitrophenolicus 98.6% 97 | S e i t e Nocardioides daeguensis 98.38% 24 Salt meadow Spirosoma 95 Spirosoma pulveris 94.52% Bad Endbach enbachense Spirosoma aerophilum 94.3% Spirosoma swuense 94.29% 25 Water fountain Novosphingobium 100 Novosphingobium panipatense 97.38% Bad Nauheim indicum Novosphingobium mathurense 97.38% Novosphingobium barchaimii 97.17% 26 Salt meadow Methyllophilus 99.57 Methylophilus quaylei 99.36% Münzenberg rhizosphaerae Methylophilus luteus 98.94% Methylophilus flavus 98.72% 27 Salt meadow Sphyngopyxis 100 Sphingopyxis chilensis 98.2% Münzenberg taejonensis Sphingopyxis ginsengisoli 98.2% Sphingopyxis italic 98.10 % 28 Salt meadow Pseudomonas 100 Pseudomonas hunanensis 99.79% Münzenberg putida Pseudomonas monteilii 99.79% Pseudomonas taiwanensis 99.69% 29 Salt meadow Pseudomonas 100 Pseudomonas hunanensis 99.79% Münzenberg hunanensis Pseudomonas monteilii 99.79% Pseudomonas taiwanensis 99.69% 30 Salt meadow Pseudomonas 100 Pseudomonas hunanensis 99.79% Münzenberg putida Pseudomonas monteilii 99.79% Pseudomonas taiwanensis 99.69% 31 Salt meadow Pseudomonas 100 Pseudomonas hunanensis 99.79% Münzenberg putida Pseudomonas monteilii 99.79% Pseudomonas taiwanensis 99.69% Table 1. Bacterial isolates obtained from different salty habitats and their next relatives based on the 16S rRNA sequence. As showed in table 1. most of the colonies were isolated from salt meadows and the genera with high prevalence in the samples were Ancylobacter, Pseudomonas, Variovorax, Paenibacillus and Hyphomicrobium, moreover the most predominant morphology was rod-shaped (Table S.1). 98 | S e i t e After taxonomic identification, the isolates were grown in FWM liquid medium with methanol and lanthanum addition. For strain 14 (99.25% similarity to Idonella dechlorotans) and strain 15 (99.05% similarity to Aurantimonas coralicida) a high level of turbidity (measured as optical density) and cell numbers (observed microscopically) were determined. A growth curve on methanol for these two strains was determined in presence and absence of La3+ as depicted in Fig.1 0.3 14+MeOH 14+MeOH+La 0.2 15+MeOH 15+MeOH+La Control 0.1 0.0 0 1 2 3 4 5 6 Time (Days) Figure 1. Growth of strains 14 and 15 in freshwater media with La3+ (LaCl3 30 µM) and MeOH (Methanol 5%v/v). Control is medium without bacteria addition. Values represent the average of eight replicate growth experiments with corresponding standard deviations, the optical density (OD 600 ) was used as growth parameter. As shown in the Fig. 1, no remarkable difference in growth of both strains on methanol with or without lanthanum was observed. Therefore, lanthanum did not allow better growth on methanol. With strain 15 growth was slightly stimulated in the presence of lanthanum although it was not significant different to growth in absence of lanthanum. DISCUSSION Different samples from naturally salty environments within Hesse, Germany that included soils, water and branches impregnated with saline water were used for this study. These samples were used as inoculum for enrichment cultures in freshwater medium with methanol in presence of lanthanum. After three subsequent transfers in liquid medium the enriched bacteria were plated on FWM 99 | S e i t e OD600 agar medium. A great variety of colonies and bacterial cell morphologies checked by microscopy were obtained. DNA was extracted from thirty-one pure colonies and based on the 16S rRNA gene sequence a taxonomic affiliation revealed that 83.0% of the isolates belong to the phylum Proteobacteria, 7.0% to Firmicutes, 7.0% to Actinobacteria and 3.0% to Bacteroidetes. Several isolates were affiliated to genera Ancylobacter, Pseudomonas, Variovorax, Paenibacillus and Hyphomicrobium. For genus Ancylobacter methanol consumption was demonstrated and the presence of mxaF-gene that encodes a methanol dehydrogenase dependent of calcium as showed for Ancylobacter aquaticus (Lau et al., 2013) and Ancylobacter sonchi (Agafonova et al., 2017). However also the presence of xoxF-gene a methanol dehydrogenase dependent of lanthanum has been reported for Ancylobacter sp. FA202 and Ancylobacter sp. 501b. (Huang et al., 2018). Pseudomonas was considered a non-methylotrophic model organism but recently a lanthanide-dependent PQQ-alcohol dehydrogenase (PedH) was discovered, which is a homologue of the xoxF-protein which has enzymatic activity on aldehydes, aromatic primary and secondary alcohols and is dependent of lanthanides such as La3+, Ce3+, Pr3+, Sm3+(Wehrmann et al., 2017). Variovorax has been described as a genus of versatile metabolism with abilities as plant growth promoters (S.-L. Sun et al., 2018); a metagenomic study to identify the diversity, abundance and activity of methylotrophs revealed a great abundance of Variovorax in the methanol-enriched pea rhizosphere community (Macey et al., 2020); Paenibacillus species can be plant associated and have a good ability to fix nitrogen (Hong et al., 2009), the capacity to use methanol in this genus has been also described (Madhaiyan et al., 2016). The genus Hyphomicrobium is known as facultative methylotrophs which can be found in water or soil (Martineau et al., 2013), methanol dehydrogenase genes such as mxaF are used as molecular markers for identification of new species of Hyphomicrobium (Fesefeldt and Gliesche, 1997) but also xoxF-gene has been detected (Huang et al., 2018). To evaluate the need of lanthanum for methanol consumption, lanthanum was added to freshwater medium and growth of strain 14 (99.25% similarity to Idonella dechlorotans) and strain 15 (99.05% similarity to Aurantimonas coralicida) was determined. Growth was very similar in presence and absence of lanthanum (Fig. 1). Similar results have been reported for growth of Methylobacterium extorquens in studies with methanol with and without lanthanum (Good et al., 2019; Vu et al., 2016). Also growth of Methylosinus trichosporium in medium with methanol and 100 | S e i t e cerium was not reported to be different than with methanol (Ul Haque et al., 2015). Moreover OD values obtained for strain 14 and 15 are lower (OD600 0.1-0.2) in comparison to the studies mentioned before where values were around OD600 0.4- 1). The low yield exhibited for both strains may indicate other specific methylotrophy metabolic modules including the serine cycle, the ethylmalonyl- CoA-pathway EMCP, the H4MPT-linked pathway and not the presence of methanol dehydrogenases encoded by mxaF and xoxF genes as described by Chistoserdova, (2011) for which other factors may affect the yield growth. This study revealed a great variety of different bacterial genera able to grow on methanol. No clear indication for growth using the lanthanum-dependent methanol dehydrogenase was found. A better identification of lanthanum-dependent methylotrophic bacteria in enrichment cultures could be the detection of xoxF-gene through molecular probes in realtime PCR assays or dot plot analysis. 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A., DiSpirito, A. A., & Semrau, J. D. (2015). Cerium regulates expression of alternative methanol dehydrogenases in Methylosinus trichosporium OB3b. Applied and Environmental Microbiology, 81(21), 7546–7552. https://doi.org/10.1128/AEM.02542-15 Vu, H. N., Subuyuj, G. A., Vijayakumar, S., Good, N. M., Martinez-Gomez, N. C., & Skovran, E. (2016). Lanthanide-dependent regulation of methanol oxidation systems in Methylobacterium extorquens AM1 and their contribution to methanol growth. Journal of Bacteriology, 198(8), 1250– 1259. https://doi.org/10.1128/JB.00937-15 Wehrmann, M., Billard, P., Martin-Meriadec, A., Zegeye, A., & Klebensberger, J. (2017). Functional role of lanthanides in enzymatic activity and transcriptional regulation of pyrroloquinoline quinone-dependent alcohol dehydrogenases in Pseudomonas putida KT2440. MBio, 8(3), 570–587. https://doi.org/10.1128/mBio.00570-17 Widdel, F., & Bak, F. (1992). Gram-Negative Mesophilic Sulfate-Reducing Bacteria. In The Prokaryotes (pp. 3352–3378). Springer New York. https://doi.org/10.1007/978-1-4757-2191-1_21 Yoon, S.-H., Ha, S.-M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology, 67(5), 1613–1617. https://doi.org/10.1099/ijsem.0.001755 103 | S e i t e Supplementary Data - Screening of bacterial strains with methylotrophic activity lanthanum dependent Table S1. Bacterial isolates obtained from different salty habitats with their morphologies and colony shapes Strain N° Source Blast Bacteria 16S Colony shape Morphology and color 1 Salt meadow Bad Ancylobacter defluvii Round cream Rod-shaped Endbach 2 Salt meadow Bad Ancylobacter defluvii Round White Rod-shaped Endbach 3 Salt meadow Paenibacillus Irregular Rod-shaped Bad Endbach glucanolyticus yellow 4 Salt meadow Rhizobium nepotum Round Rod-shaped Bad Endbach transparent 5 Salt meadow Ancylobacter defluvii Round white Rod-shaped Bad Endbach 6 Salt meadow Variovorax Round red Rod-shaped Münzenberg boronicumulans 7 Salt meadow Paenibacillus validus Irregular white long curved rod Münzenberg 8 Graduation Ancylobacter Round cream Rod-shaped Building rudongensis Bad Nauheim 9 Graduation Jiella aquimaris Round cream Rod-shaped Building Bad Nauheim 10 Graduation Tistrella bauzanensis Round white Rod-shaped building Bad Nauheim 11 Water Fountain Paracoccus homiensis Round Coccus Bad Nauheim transparent 12 Salt meadow Azospirillum oryzae Round cream Vibrioid Münzenberg 104 | S e i t e 13 Salt meadow Pseudomonas Round green Rod-shaped Münzenberg hunanensis 14 Salt meadow Idonella dechloratans Round Rod-shaped Münzenberg transparent 15 Graduation building Aurantimonas coralicida Round yellow Rod-shaped Bad Nauheim 16 Salt meadow Hyphomicrobium facile Round white Rod-shaped with prosteca Bad Endbach subsp. facile 18 Salt meadow Variovorax Round white Rod-shaped Bad Endbach boronicumulans 20 Salt meadow Hyphomicrobium faciles Round white Rod-shaped with prosteca Bad Endbach subs. facile 21 Salt meadow Microbacterium Round cream Vibrioid Bad Endbach natoriense 22 Salt meadow Variovorax Round blue Rod-shaped Bad Endbach boronicumulans 23 Salt meadow Nocardiodes simplex Irregular Rod-shaped Bad Endbach transparent 24 Salt meadow Spirosoma Round cream Rod-shaped Bad Endbach enbachense 25 Water fountain Novosphingobium Round blue Long curved rod Bad Nauheim indicum 26 Salt meadow Methyllophilus Irregular Rod-shaped Münzenberg rhizosphaerae cream 27 Salt meadow Sphyngopyxis Round cream Rod-shaped Münzenberg taejonensis 28 Salt meadow Pseudomonas Round Rod-shaped Münzenberg putida transparent 29 Salt meadow Pseudomonas Round yellow Rod-shaped Münzenberg hunanensis 30 Salt meadow Pseudomonas Round cream Rod-shaped Münzenberg putida 31 Salt meadow Pseudomonas Round white Rod-shaped Münzenberg putida 105 | S e i t e CHAPTER 6. GENERAL DISCUSSION 106 | S e i t e GENERAL DISCUSSION Recent studies have shown that rare metals play an important role to a specialized group of bacteria involved in the global carbon cycle. Rare metals such as lanthanum and cesium are required for the activity of a widespread methanol dehydrogenase (MDH) enzyme encoded by the xoxF-gene which is used for methanol utilization as source of carbon and energy by some bacteria. Genes encoding for methanol dehydrogenase have been detected in rhizosphere of pea plants, rice, cereals and grasses (Butterfield et al., 2016; Knief et al., 2012; Tsurumaru et al., 2015) and some of them affiliated to xoxF, however the role of this gene in plant root-bacteria interaction is unclear. Hartmannibacter diazotrophicus E19T was isolated from a saline soil and exhibited abilities as plant growth promoter including ACC-deaminase production, nitrogen fixation, phosphorus solubilization (Suarez et al., 2015) but also methylotrophic activity as methanol consumer in presence of lanthanum associated to the presence of xoxF- gene was evidenced (Lv et al., 2017). Methylotrophic activity assays In order to reassess the methylotrophic activity of H. diazotrophicus E19T in liquid culture, a growth curve in a mineral medium with methanol 5% v/v and 30 µM lanthanum was determined and methylotrophic growth via lanthanum-dependent MDH was confirmed. The suitable concentration of lanthanum for optimal growth of H. diazotrophicus E19T was determined in growth studies with the pure culture, however, no significant differences were founded for lanthanum concentrations evaluated (Chapter 2). The effect of lanthanum on growth with methanol of Methylacidiphilum fumariolicum SolV was described by Pol et al., (2014) to be essential, however in contrast to the study of Pol et al., (2014) in this study no differences in the growth yield were found when concentrations of lanthanum varied. Greenhouse trials To evaluate the plant response under salt stress to inoculation of H. diazotrophicus E19T and lanthanum supplementation, a pot experiment with barley plants in greenhouse was carried out (Chapter 2). The trials of the experiment comprised 107 | S e i t e barley seeds inoculated with strain E19T sowed in saline soil with/without lanthanum, barley seeds inoculated with strain E19T dead biomass sowed in saline soil with/without lanthanum, barley seeds in saline soil with lanthanum and the control with barley seeds in saline soil. Growth of plants was monitored after thirty days, when shoots and roots were harvested, and the fresh and dry weights were determined. Furthermore, plant samples from roots and rhizosphere soil were taken to explore changes in microbial diversity upon the treatments and to look into colonization of roots with E19T. The barley plants biomass of shoots and roots revealed no effect or differences between plants inoculated with H. diazotrophicus E19T with and without lanthanum addition however better biomass was obtained in comparison to the treatments with dead biomass and the non-inoculated control. The results demonstrated that strain E19T can promote growth of barley plants in salty conditions which is in agreement with the study of Suarez et al., (2015). These authors postulated a mechanism of H. diazotrophicus E19T to reduce salt stress based on the lowering of ethylene levels in the plant by bacterial production of ACC deaminase. Unexpectedly with lanthanum addition to soil without bacterial inoculation the plant obtained as good biomass values as the treatments with seed inoculation. The effect of lanthanum to increase plant tolerance under salty conditions was discussed to origin from mechanisms such as up-regulation of the antioxidant capacity in the chloroplast through ascorbate-glutathione cycle, the rising of cytoplasmic calcium levels which block the reactive oxygen species (ROS) production and the enhancement of chlorophyll and carotenoids contents (Huang et al., 2018; Li et al., 2007; Liu et al., 2016). The inoculation of bacteria with plant promotion activity may also change the composition of rhizosphere communities thus indirectly affecting plant growth (Gadhave et al., 2018; Wang et al., 2018; Zhang et al., 2019; Zhang et al., 2019). In the current study, the indicators for microbial diversity were evaluated comparing effects of H. diazotrophicus inoculation with those of lanthanum supplementation. In roots and rhizosphere soil alpha diversity indices showed a significant increase in species richness in treatments of plants with H. diazotrophicus and H. diazotrophicus with lanthanum compared to treatments with death biomass and lanthanum alone. According to nonmetric multidimensional scaling (NMDS) most similar communities were found in treatments of H. 108 | S e i t e diazotrophicus and H. diazotrophicus with La; most different was the community in control soil (Chapter 2). The quantification of H. diazotrophicus E19T colonization in rhizosphere soil and root environments was explored through qPCR using a specifically designed primer set targeting the 16S RNA gene sequence. High DNA copy numbers of strain E19T were detected in barley roots (5.3 × 107 DNA copy number g-1 root dry weight) 30 days after seed inoculation. In rhizosphere soil strain E19T DNA copy number were 3.2 × 104 DNA copy number g-1 soil dry weight which indicates the good bacterial competence for the plant root colonization but also that the rhizosphere is a suitable environment for the bacteria. The quantification of bacterial cell number in root samples of plants grown in pots by qPCR are in similar values reported for studies with DNA quantification of other PGPR species; plants of Brassica oleracea were inoculated with Enterobacter radicincitans resulting in concentrations of 108 bacterial cells g-1 root fresh weight (Ruppel et al., 2006), in the same way, quantification of Azospirillum lipoferum CRT1 inoculated in maize plants in roots was 104–106 DNA copy number g-1 of root dry weight (Couillerot et al., 2010). On the other hand, the presence of lanthanum not increased the cell density of strain E19T, the treatments with seed inoculation of H. diazotrophicus and H. diazotrophicus with lanthanum exhibited similar densities for the inoculated bacteria on roots, while in rhizosphere soil the number of DNA copies were higher in the treatment of strain E19T without lanthanum supplementation. The differences in cell numbers of H. diazotrophicus in roots and rhizosphere soil might be due to the fact that during root growth and cell-plant division with pectin biosynthesis the methanol is directly available for H. diazotrophicus in the root and on its surface whereas in rhizosphere the exudate methanol is consumed by a great diversity of bacterial groups. The high substrate competition in the rhizosphere (Sy et al., 2005) may explain the lower cell numbers of H. diazotrophicus in rhizosphere. For the treatment with H. diazotrophicus and lanthanum supplementation the growth response of H. diazotrophicus may mostly be conditioned to substrate and lanthanum sensing, the presence of a “lanthanide- switch” which is described as transcriptional response can at low concentrations of lanthanum upregulate the xoxF activity conditioning the uptake of methanol and downregulate other metabolic routes for consumption of other carbon sources. A study carried out by Wehrmann et al., (2020) described the growth of 109 | S e i t e Pseudomonas putida containing PedH-MDH lanthanum dependent, a homologue of xoxF-MDH in presence of 10 µM lanthanum and other carbon sources; a downregulation of metabolic proteins expression was observed when growth occurred with glucose and lanthanum. The quantification of H. diazotrophicus E19T in roots also was performed using a designed primer set targeting the xoxF-gene sequence and 4.8 x 106 xoxF copies numbers g-1 root dry weight were obtained, which is similar to those DNA copy numbers retrieved with the specific 16S RNA gene assay (Chapter 2). These results confirm the colonization of H. diazotrophicus in roots and on the root surface since the primer was designed specifically for xoxF-gene sequence of H. diazotrophicus. The designed xoxF primer will open the path to study the expression of xoxF gene on RNA samples for future research focused on bacteria plant interactions. Bioprospection For this study, halotolerant methanol-oxidizing and lanthanum-dependent bacteria from salt-affected environmental samples (soil, water, graduation building) have been enriched in a mineral medium supplemented with methanol 5% v/v and 30 µM lanthanum, 31 isolates were recovered and by 16S RNA gene sequencing the taxonomic affiliation revealed most of them belonging to phylums Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes, and some of them also have been reported with genes associated to methylotrophic activity dependent of rare metals (Chapter 5, Table 1). The isolates 14 and 15 exhibited good growth in liquid medium with methanol and lanthanum. Therefor a growth curve in liquid medium with methanol with/without lanthanum supplementation was determined which revealed that lanthanum not caused significant difference in the growth of both strains. However methylotrophic bacteria have high metabolic flexibility, different carbon assimilation pathways have been reported for energy conservation required in growth process including serine cycle, RuMP cycle, Benson cycle where lanthanides are not involved (Khmelenina et al., 2019). Moreover, from the enrichment and screening from soil samples (Chapter 5, Table 1), the bacterium 24 designated I-24 had a 95.0% 16S rRNA gene similarity to the next relative. Since a 16S rRNA gene identity value < 98.7% represents a new species a polyphasic approach was carried out for detailed characterization and identification of isolate I-24. The polyphasic study revealed that isolate I-24 was aerobic, non- motile, rod-shaped, catalase-positive, oxidase-positive and grew optimally at pH 110 | S e i t e 7 and 25 °C. Based on 16S rRNA gene sequence close relatives are Spirosoma agri and Spirosoma terrae and isolate I-24 was grouped into the family Spirosomaceae, genus Spirosoma and with the proposed species name Spirosoma endbachense accepted. The draft genome sequence of Spirosoma endbachense has a size of 10,326,072 bp with a G+C of 47.7% and the completeness value was >99%. Secondary metabolite clusters annotated for Spirosoma endbachense were ladderane, terpene, polyketide synthase type I and III (T1PKS, T3PKS) and non- ribosomal peptide synthetase (NRPS) (Chapter 3). For close the relatives Spirosoma agri S7-3-3 (KCTC 52727) and Spirosoma terrae 15J9-4 (KCTC 52035) a genome sequencing was not available. For detailed taxonomic characterization whole genome sequencing techniques are suggested to be used along with a polyphasic study to provide more information about identification, phylogenetic analysis, metabolic pathways, antimicrobial resistance and enable comparative genomic. Therefor the total genomes from both strains and S. endbachense were sequenced using a MiSeqv3 sequencer system (Illumina), assembled and annotated. The draft genome sequence of Spirosoma agri has a size of 7,239,915 bp with a G+C content of 50.6% while for Spirosoma terrae the size is of 7,551,610 bp with a G+C content of 47.3% (Chapter 4). Both genomes are ≥ 99% complete. Both species have genes for alkaline phosphatase, cellulose and amylase activity and many other carbohydrate-active enzymes. Furthermore gene clusters for the production of ladderane, terpen, polyketide synthase, non-ribolsomal peptide synthesis and secondary metabolite biosynthesis were annotated. No reports have so far focused on the attempt to characterize methanol utilizing bacteria containing the recently discovered xoxF-MDH from saline and alkaline environments. Therefor in an isolation campaign methylotrophic bacteria from saline environments have been enriched in the presence of lanthanum to possibly retrieve halotolerant bacteria which consume methanol via xoxF-MDH. These isolates are potential new candidates for plant growth promotion under salt stress due to their adaptation to natural salt environments. Other methylotrophic bacteria have been reported to mitigate salt stress effects on plants like the halotolerant Actinobacterium Nocardioides sp. NIMMe6 which was reported to enhance the growth of wheat plants under salt stress (Meena et al., 2020). For the methanol- 111 | S e i t e utilising Bacillus methylotrophicus M4-1 enhanced uptake of Mg2+, K+ was reported and the ability to reduce Na+ content in leaves of winter wheat under salt stress (Ji et al., 2020). For the latter bacteria however pathways or genes involved in C1 compounds have not been elucidated. 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Thank you very much. I would also like to extend my greatest gratitude to Dr. Stefan Ratering for his great feedback, knowledge sharing, excellent encouragement, and guidance thanks a lot. I am deeply grateful to Prof. Hans Koyro Institute of Plant Ecology, IFZ, Justus- Liebig University Gießen for his interest and disposition as second supervisor. Very special thanks to Rita Geißler-Plaum who always encourage and supporting me during the work in lab and the greenhouse and thanks very much for your patience and friendly help. I also wish to thanks to Belinda Schneider for her support in molecular lab and thanks to make the atmosphere of work funnier. Many thanks to Renate Baumann for her helpful advice and practical suggestions with the work of lab. Special thanks to Colombian-Ecuadorian group David Rosado, Christian Suarez Santiago Quiroga, Angel Franco and Yina Cifuentes for your listening, support, advise, friendship and being there always when I needed. I also had the great pleasure of working with Alessandra Dupont, Julia Sacharow, Hülya Kaplan, Corina Maisinger, Rommy Auerbach, Yulduz Abdullaeva, Binoy Ambika Manirajan, whom I thank for their collaboration and friendship. I gratefully acknowledge the support and help from the staff and technicians of the Institute of Applied Microbiology Corina, Gundi, Katja, Maria, Jan, and Monika. Most importantly, I would also like to thank my parents and brothers for their unfailing support and continuous encouragement whenever I have needed. 115 | S e i t e