This is the accepted version of the journal article: Atanasov, Kostadin E.; Miñana-Galbis, David; Gallego, Julia; [et al.]. «Pseu- domonas germanica sp. nov., isolated from Iris germanica rhizomes». Interna- tional Journal of Systematic and Evolutionary Microbiology, Vol. 72, Issue 3 (March 2022). DOI 10.1099/ijsem.0.005268 This version is available at https://ddd.uab.cat/record/257481 under the terms of the license 1 Pseudomonas germanica sp. nov., isolated from Iris 2 germanica rhizomes 3 4 Kostadin Evgeniev Atanasov1,2, David Miñana-Galbis3, Julia Gallego4, Annabel Serpico4, 5 Montserrat Bosch4, Teresa Altabella1,2 and Albert Ferrer1,5 6 1 Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, Barcelona, 7 Spain. 8 2 Department of Biology, Healthcare and the Environment, Plant Physiology Section, Faculty 9 of Pharmacy and Food Sciences, University of Barcelona, Spain. 10 3 Department of Biology, Healthcare and the Environment, Microbiology Section, Faculty of 11 Pharmacy and Food Sciences, University of Barcelona, Spain. 12 4 Applied Microbiology and Biotechnology Unit, LEITAT Technological Center, Terrassa, 13 Spain. 14 5 Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, 15 University of Barcelona, Spain. 16 Corresponding author: Kostadin E. Atanasov 17 The email address for the corresponding author: kostadin.atanasov@cragenomica.es 18 Keywords: Pseudomonas, Iris germanica, endophyte, multilocus sequence analysis, type- 19 strain, average nucleotide identity, digital DNA-DNA hybridization. 20 Repositories: 21 BioProject: PRJNA705867; BioSample: SAMN18105273; Sequence Read Archive (SRA): 22 SRR14429882; Assembly: ASM1961465v1; GenBank: MZ758888.1. 23 1 24 ABSTRACT 25 Through bacterial plant-endophyte extraction from rhizomes of Iris germanica plant, a Gram- 26 negative, aerobic, catalase and oxidase positive gammaproteobacterial referred to as FIT28T, 27 was isolated. Strain FIT28T shows vigorous growth on nutrient rich media within the 28 temperature ranging from 4 to 35 ºC, with optimal growth at 28 ºC, a wide pH adaptation from 29 pH 5 to 11, and salt tolerance up to 6% (w/v) NaCl. Colonies are white-yellow and quickly 30 become mucoid. Analysis of 16S rRNA gene sequence placed the strain within the 31 Pseudomonas genus, and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, gyrB 32 and rpoD concatenated sequence revealed that the closest relatives of FIT28T are Pseudomonas 33 zeae OE48.2T, Pseudomonas crudilactis UCMA 17988T, Pseudomonas tensinigenes ZA5.3T, 34 Pseudomonas helmanticensis OHA11T, Pseudomonas baetica a390T, Pseudomonas iridis 35 P42T, Pseudomonas atagonensis PS14T, and Pseudomonas koreensis Ps 9-14T, within the 36 Pseudomonas koreensis subgroup of the Pseudomonas fluorescens lineage. Strain FIT28T 37 genome size is about 6.7 Mb with 59.09% GC content. Average nucleotide identity (ANI) and 38 digital DNA-DNA hybridization (dDDH) values calculated from the genomic sequences of 39 FIT28T, and the closely related P. zeae OE48.2 type-species are 95.23% and 63.4%, 40 respectively. Biochemical, metabolic, and chemotaxonomic studies further support our 41 proposal that Pseudomonas germanica sp. nov., should be considered a novel species of the 42 genus Pseudomonas. Hence, the type strain FIT28T (= LMG 32353T = DSM 112698T) was 43 deposited in public cell-type culture centers. 44 2 45 Introduction 46 Pseudomonas Migula 1894 is a widely distributed genus of rod-shaped gram-negative 47 gammaproteobacteria with monotrichous or lophotrichous flagella. These bacteria are aerobic 48 and have catalase and cytochrome C oxidase activity. Furthermore, they exhibit good growth 49 in nutrient-rich media, salt tolerance, and growth within a range of pH (between 5 and 10). 50 Niches where Pseudomonas can be found are diverse and include environmental soil samples, 51 hot spring water, river and lake samples, wastewater, and in association with living organisms. 52 Up to date, the genus contains 270 published species with accepted correct names 53 (www.bacterio.net). Because Pseudomonas is one of the most complex genera, during last 54 decades different genetic and genome-based taxonomic methods have been tested in order to 55 identify and classify taxonomically novel isolated Pseudomonas species. Multilocus sequence 56 analysis (MLSA) is a gene-based technique where 16S rRNA gene sequence is combined with 57 other protein-coding housekeeping genes such as rpoB, gyrB and rpoD, resulting in a unique 58 concatenated large sequence which is aligned to other species in order to investigate 59 phylogenetic relationship. In 2018, Peix et al., conducted MLSA study on Pseudomonas 60 species and outputted a phylogenetic analysis describing species clustering in lineages and 61 groups. Authors suggested the existence of P. fluorescens lineage, P. aeruginosa lineage, and 62 P. pertucinogena lineage. Among them, the groups integrating the most species-abundant P. 63 fluorescens lineage were P. fluorescens, P. lutea, P. syringae, P. rhizosphaerae, P. putida, P. 64 anguilliseptica, and P. straminea group [1]. Nowadays, the increasing number of fully 65 sequenced genomes enable in silico global genome comparisons analysis. Comparative 66 genomics of different species is demonstrating higher accuracy in species-species delineation 67 and is prompting to review and reclassify some taxon species initially considered different but 68 belonging to the same species or subspecies. Pan-genome tools for in silico analysis algorithms 69 are average nucleotide identity (ANI), orthologous ANI (orthoANI), blast-based ANI (ANIb), 3 70 MUMer3-based ANI (ANIm), USEARCH-based ANI (ANIu), and digital DNA-DNA 71 hybridization (dDDH). ANI and dDDH analysis fix species boundaries percentages at 95 – 72 96% and 70%, respectively [2]. Hesse et al. (2018) compared genomes of Pseudomonas type 73 strains and subspecies by conducting ANI analysis and constructing phylogenetic relationship 74 based on the alignment of protein sequence of orthologous genes. Authors were able to relocate 75 strains from previous MLSA studies [3, 4] and reported subdivision of the P. fluorescens group 76 in ten subgroups composed by P. fragi, P. asplenii, P. gessardii, P. fluorescens, P. protegenes, 77 P. chlororaphis, P. corrugata, P. koreensis, P. jessenii, and P. mandelii [5]. Nevertheless, 78 despite of the power of gene and genome based-analysis, borderline species identification and 79 delineation are still difficult and can require a combination of exhaustive biochemical, 80 physicochemical, and molecular characterization [6]. 81 Species of the P. fluorescens group are soil-born bacteria that are highly associated with plants 82 organisms. However, increasing data are suggesting that some species could infect animals 83 and/or produce infections [7]. Relationship with the plant host can be neutral, pathogenic, or 84 even beneficial by stimulating plant growth and development through diverse molecular 85 mechanism [8]. Pseudomonades are capable to synthetize a broad spectrum of specialized 86 bioactive metabolites with antimicrobial, antioxidant, anticancer and antifungal activities, 87 among others [9], which makes them interesting as a potential new source of this kind of 88 compounds. Hereafter we describe the characterization of a new Pseudomonas species isolated 89 from Iris germanica rhizomes. Combination of whole genome sequencing data with 90 biochemical and physicochemical analyses enabled us to propose Pseudomonas germanica 91 strain FIT28T as a new type-strain. 92 Isolation and Ecology 93 Strain FIT28T was isolated from the endosphere of Iris germanica rhizomes at the Marimurtra 94 Botanical Garden of Blanes in Catalonia (Spain). Entire plants were collected and examined 4 95 for pathogenic lesions, injuries, or the presence of active infections. Only fully healthy plants 96 were used for endophytes extraction. Soil attached to I. germanica rhizomes was first washed 97 off with sterile distilled water and next with phosphate buffered saline (PBS; 1.8 mM KH2PO4, 98 10 mM Na2HPO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4). Surface sterilization was performed 99 by washing rhizomes with 70% ethanol for 5 min, 2% (w/v) sodium hypochlorite for 10 min, 100 and 70% ethanol for 45 min. Rhizomes were then washed up to seven times with sterile- 101 distilled water. Sterilized rhizomes were dried with paper towel under sterile conditions, 102 transferred to a mortar, and homogenized with pestle in PBS solution. A serial dilution of the 103 crude extract was prepared and 50 µl plated on Tryptic Soy Agar medium (TSA, Sigma- 104 Aldrich, St Louis, USA). Plates were incubated at 30 ºC for 15 days and individual colonies 105 were transferred on new TSA plates and spreaded by extenuation. A single colony was 106 inoculated into 5 ml Tryptic Soy Broth (TSB, Becton Dickinson, Franklin Lakes, USA) 107 medium and incubated for 24h – 72h at room temperature. Endophyte culture was mixed with 108 glycerol up to 25% (v/v) final concentration and stored at -80 ºC. 109 Multilocus Sequence Analysis (MLSA) and Genome Features 110 A single colony grown on TSA was transferred to TSB medium and incubated for 16h at 28 111 ºC, with constant shaking at 190 rpm. Cells were pelleted and DNA extracted by a modified 112 CTAB protocol [10, 11]. For the phylogenetic genus location of strain FIT28T, PCR 113 amplification of 16S rRNA V5/V6/V7 regions was carried out with 799F (5'- 114 AACMGGATTAGATACCCKG-3') and 1193R (5'-ACGTCATCCCCACCTTCC-3') primers 115 [12]. Five microliters of the PCR reaction were treated with ExoSAP-IT (Thermo Fisher 116 Scientific, Waltham, USA) and sequenced for triplicate. A consensus sequence was generated 117 and a preliminary taxonomic location of strain FIT28T stablished by nucleotide-blast analysis 118 against the bacterial 16S rRNA database of NCBI and the Ribosomal Database Project (RDP; 119 http://rdp.cme.msu.edu/) [13]. 5 120 For high-quality genomic DNA isolation, 20 ml of a strain FIT28T culture in TSB was used. 121 DNA extraction was performed with Wizard Genomic DNA Extraction Kit (Promega, 122 Madison, USA). Two hundred microliters of genomic crude extract were treated for 30 min 123 with 3 µl of 10 mg·ml-1 RNase A DNase free (Panreac AppliChem, Darmstadt, Germany), and 124 then deproteinized by chloroform-isoamyl alcohol treatment, followed by a 70% ethanol wash 125 and resuspended in 300 µl of nuclease free water. DNA was passed through Genomic DNA 126 Clean & Concentrator-10 column (Zymo Research, Irvin, USA) and eluted in TE buffer 127 according to manufacturer instructions. DNA was quantified by Qubit 3.0 fluorometer (Life 128 Technologies, Carlsbad, USA), and the DNA integrity number (DIN) was assessed with 129 Bioanalyzer DNA 12000 chip (Agilent, Santa Clara, USA). Whole genome sequencing of 130 strain FIT28T was performed with Illumina Novaseq (2x150 bp) and PacBio RSII platforms. 131 Achieved genome coverage was 495 times. Illumina and PacBio sequencing reads were used 132 to construct a hybrid genome assembly using Unicycler v0.4.8 [14]. 133 Genome annotation was performed with the Prodigal pipeline in order to identify protein 134 coding genes [15]. Genome contamination was screened with ContEst16S [16]. Ribosomal 16S 135 rRNA genes were predicted on assembled contigs using Barnnap 136 (https://github.com/tseemann/barrnap). A consensus sequence of the ribosomal 16S RNA gene 137 of strain FIT28T was extracted and analyzed with the taxonomically united database of 138 EzBioCloud [17]. Search was limited to bacterial type strains and genome assemblies of related 139 species downloaded from GenBank database. For MLSA we used sequences of the 140 housekeeping rpoB, gyrB and rpoD genes (Table S1) [18]. Sequences of 16S rRNA, rpoB, 141 gyrB, and rpoD were extracted from genome assemblies, concatenated, and aligned with 142 MUSCLE algorithm [19]. Genome assemblies for P. defluvii WHCP16T and P. 143 turukhanskensis IB1.1T were not available at the moment of writing this paper. For P. 144 helmanticensis OHA11T (= CECT 8548T = LMG 28168T) raw genome assembly was 6 145 downloaded from the JGI Genome Portal [20]. Genomes of P. helmanticensis OHA11T, P. zeae 146 OE 48.2T, P. tensinigenes ZA 5.3T, P. iridis P42T, and P. hamedanensis SWRI65T, were 147 annotated with PATRIC 3.6.9 [21] and sequences for MLSA extracted from gene annotation. 148 A total number of 41 species were considered for the study. We used P. aeruginosa DSM 149 50071T as a rooting outgroup. Neighbor-Joining trees were generated with Mega 10 software 150 [22]. Afterwards, distance matrices were calculated by the Jukes-Cantor method and bootstrap 151 analysis was performed based on 1000 re-sampling [23]. Gaps or missing data were treated by 152 pairwise deletion. 153 Comparison of 16S rRNA phylogenetic analysis located strain FIT28T with P. zeae OE48.2T 154 (99.87%), P. tensinigenes ZA5.3T (99.87%), P. helmanticensis OHA11T (99.86%), P. 155 hamedanensis SWRI65T (99.67%), P. crudilactis UCMA 17988T (99.61%), P. atagonensis 156 PS14T (99.59%), P. baetica a390T (99.52%), and P. iridis P42T (Fig. S1.1). Nucleotide-blast 157 and pairwise alignment (https://blast.ncbi.nlm.nih.gov/) of the concatenated housekeeping 158 genes outputted next sequence identities: 98.98% (P. zeae OE48.2T), 98.91% (P. tensinigenes 159 ZA5.3T), 96.96% (P. helmanticensis OHA11T), 97.52% (P. hamedanensis SWRI65T), 98.50% 160 (P. crudilactis UCMA 17988T), 97.24% (P. atagonensis PS14T), 96.10% (P. baetica a390T), 161 and 96.27% (P. iridis P42T)(Table S2). Similar results were obtained when we included 162 additional housekeeping genes corA, atpD and recA [24]. Concatenated sequence of 16S 163 rRNA, rpoB, gyrB, rpoD, corA, atpD and recA genes were aligned with 98.98% of sequence 164 identity, 92% of coverage, between FIT28T and P. zeae OE48.2T. 98.44% identity was 165 observed when 16S rRNA, corA, atpD and recA where used, but in this case, sequence 166 coverage dropped down to 79%. Furthermore, up-to-date bacterial core gene (UBCG) [25] 167 analysis was carried out by aligning 91 concatenated core genes where 50 out of UBCGs 168 supported FIT28T and P. zeae OE48.2T phylogenetic relationship with 98.01% of sequence 7 169 identity (Fig. S1.2). These values were slightly above the threshold of 97% purposed in MLSA 170 for the species boundary [3, 24, 26]. 171 Nevertheless, MLSA based phylogenetic-tree helped to define a thirteen-member clade formed 172 by strains FIT28T, P. zeae OE48.2T, P. crudilactis UCMA 17988T, P. tensinigenes ZA5.3T, P. 173 helmanticensis LMG 28168T (= OHA11T), P. baetica LMG 25716T (= a390T), P. iridis P42T, 174 P. atagonensis PS14T, P. koreensis DSM 16610T, P. granadensis LMG 27940T, P. 175 hamedanensis SWRI65T, P. atacamensis M7DT, and P. moravienis BS3668T (Figure 1) which 176 was considered in order to define inter-strain comparisons. Hence, in concordance to the 177 previous phylogenetic data we propose that strain FIT28T can represent a new member of the 178 P. koreensis subgroup within the P. fluorescence group of the homonym lineage. 179 In silico prediction of GC content reported a value of 59.09%, which is characteristic for 180 Pseudomonas fluorescens environmental strains [27]. Genome size was 6.713.530 base-pair 181 (bp), with predicted 6.022 protein coding genes [28], 19 rRNA coding genes, including 5S 182 rRNAs (7 genes), 16S rRNAs (6 genes), and 23S rRNAs (6 genes), and 75 tRNA coding genes 183 [29]. Based on MLSA analysis we selected thirteen-member clade species (Figure 1) for whole 184 genome comparison. Average nucleotide identity and orthoANI values were calculated by 185 pairwise comparison with Ortho ANI Tool [30]. Blast-based ANI (ANIb) and MUMmer3 ANI 186 (ANIm) were tested by JSpeciesWS [31], whereas USEARCH ANI (ANIu) was calculated 187 according to Yoon and colleagues [31]. Strain FIT28T exhibited ANI and orthoANI values of 188 95.23% and 95.51% (over 64% of genome coverage), respectively, to P. zeae OE48.2T (Table 189 1, Table S3). ANIu and ANIm values were close to the original ANI and OrthoANI with 190 percentages of 95.92% and 95.44%, respectively. Again, ANIb revealed an average value of 191 94.94% with genome coverage of 85.84%. Overall, ANI results remained inconclusive due to 192 values within the borderline interval for the generally recommended threshold of 95 – 96% for 193 considering same species [2, 32]. Hence, like in MLSA, genomic analysis was not enough to 8 194 fully differentiate between these two closely related species. Finally, dDDH analysis was 195 assessed with the Genome-to-Genome Distance Calculator (GGDC) from the DSMZ webtool 196 (https://ggdc-test.dsmz.de/ggdc.php#) with the BLAST+ local alignment algorithm [33]. We 197 used formula 2, which consists of the number of identities within high-scoring segment pairs 198 (HSPs) per total HSP length [34]. The recorded dDDH value for the nearest P. zeae OE48.2T 199 was 63.4% with an 95% of confidence interval of 60.5% - 66.2%, whereas other species 200 exhibited absolute dDDH values between 33.3% (P. moraviensis BS3668T) and 46.7% (P. 201 tensinigenes ZA5.3T) (Table 2). Thus, values are clearly below 70% sequence identity, which 202 is considered as the gold standard for new species delineation [2]. 203 Physiology and Chemotaxonomy 204 A colony of strain FIT28T grown on TSA medium was placed and immobilized on a 205 microscope glass slide by heat treatment. A Gram stain procedure was performed with a Gram 206 stain set according to manufacturer instructions (Condalab, Torrejón de Ardoz, Spain). Gram 207 negative stained cells were examined with Zeiss Axiophot FL microscope under bright field, 208 and image acquired with Olympus DP70 at the core facilities of the Center for Research in 209 Agricultural Genomics - CRAG (Figure S2). Moreover, a negative stain with uranyl acetate 210 was performed on strain FIT28T and cells visualized with transmission electron microscope 211 (TEM) Bioscan Gatan, JEOL 1010, at the Scientific and Technological Centers (CCiT) of the 212 University of Barcelona. Strain FIT28T possess a single polar flagellum and confirmed rod- 213 shaped cells. The estimated cell size was 2.3 µm length per 0.91 µm width (Figure 2). 214 In order to stablish optimal temperature growth conditions, strain FIT28T was cultured on TSA 215 medium at temperatures ranging from 4 to 40 ºC. Cell growth was observed from 4 to 35 ºC 216 with an optimal temperature stablished at 28 ºC, which agrees with previous reports for 217 environmental P. fluorescens species [35]. For pH tolerance, growth was recorded within the 218 range of pH 5-11 at 28 ºC with an optimal pH range from 6 to 7. Production of fluorescent 9 219 pigment was tested by culturing on King’s B medium (Duchefa Biochemie, Haarlem, the 220 Netherlands) for 24h and image acquisition under 365 nm UV light irradiation [36]. Catalase 221 activity was detected by placing drops of 3% (v/v) hydrogen peroxide solution on active 222 bacterial cell cultures. Bubbles formation was observed when applied to strain FIT28T, P. 223 helmanticensis CECT 8548T (= OHA11T), P. baetica CECT 7720T (= a390T), and P. 224 atagonensis CECT 9940T (= PS14T), thus indicating positive catalase activity as is in 225 agreement with previously described data. Oxidase activity was also tested on same species as 226 for catalase activity. Small amounts of solid-media active cell cultures were placed on paper 227 disc and drops of tetra-methyl-p-phenylenediamine dihydrochloride solution were added. Blue- 228 purple color was observed in all cases indicating cytochrome C oxidase activity. 229 Biochemical characteristics of strain FIT28T were examined with API 20E and API 20NE 230 (Biomerieux, Marcy-l'Étoile, France). Growth-dependent tests for the utilization of amino 231 acids, organic acids and carbohydrates were performed with API 50CH (Biomerieux). 232 Additionally, phenotypic fingerprinting for carbon source utilization and chemical sensitivity 233 was performed with Biolog GenIII Microplate (Biolog, Inc., Hayward, USA). All enzymatic, 234 physiological, and biochemical analyses were carried out at 30 ºC according to manufacturer 235 instructions. Test results reading and interpretation was achieved by manual method. Principal 236 biochemical activities and physiological features are summarized in Table 3. To build this 237 table, data for P. helmanticensis CECT 8548T, P. baetica CECT 7720T and P. atagonensis 238 CECT 9940T were obtained in this study whereas data for the other species were extracted from 239 literature [37–47]. Salt tolerance was also assessed up to 9% of NaCl (w/v) with a maximum 240 growth-tolerance at 6% NaCl for the isolated strain. This result was corroborated with the 241 miniaturized Biolog’s GenIII test recording growth capacity at 6% (w/v) of NaCl for strain 242 FIT28T. According to this, we found higher salt tolerance for strain FIT28T when compared to 243 P. zeae OE48.2T, P. tensinigenes ZA5.3T, P. crudilactis UCMA 179881T, P. helmanticensis 10 244 CECT 8548T P. baetica CECT 7720T, P. atagonensis CECT 9940T, P. granadensis LMG 245 27940T, P. hamedanensis SWRI65T, and P. atacamensis LMG 34516T, but similar salt 246 tolerance to P. iridis P42T, P. koreensis DSM 16610T, and P. moraviensis DSM 16007T (Table 247 3). FIT28T can utilize as carbon source compounds such as D-arabitol, D-fructose, D-saccharic 248 acid, formic acid, inosine, L-alanine, L-pyroglutamic acid, pectin, propionic acid, sucrose, 249 tween 40, dextrin, D-mannitol, glycyl-L-proline, methylpyruvate, α-hydroxybutiric acid, D- 250 trehalose, D-gluconic acid, L-lactic acid, glycerol, N-acetyl-D-glucosamine, L-histidine, D- 251 malic acid, L-serine, and bromo-succinic acid, while P. zeae OE48.2T exhibits negative or 252 uncertain results for the utilization of those compounds. We also found differences regarding 253 the utilization of D-cellobiose, D-sorbitol, gelatin, D-salicin, α-ketobutyric acid, D-glucose-6- 254 phosphate, D-fructose-6-phosphate, acetoacetic acid, N-acetyl-β-D-mannosamine, 3-methyl 255 glucose, D-turanose, gentiobiose, and stachyose. These compounds were found negative (D- 256 sorbitol and gelatin) or uncertain/weak when FIT28T was compared to P. zeae OE48.2T data 257 reported by Girard and colleagues [46]. Strain FIT28T, P. tensinigenes ZA5.3T, P. crudilactis 258 UCMA 17988T, P. helmanticensis CECT 8548T and P. baetica CECT 7720T have melibiose 259 oxidative activity whereas P. iridis P42T, P. atagonensis CECT, P. koreensis DSM 16610T and 260 P. moraviensis DSM 16007T were negative for this catabolic process. Oxidation of melibiose 261 was uncertain for P. zeae OE48.2T and P. hamedanensis SWRI65T. We also observed that 262 strain FIT28T is negative for arabinose oxidation and phenylacetic acid assimilations. 263 Enzymatic activities were screened and FIT28T exhibited gelatin hydrolysis capacity whereas 264 P. zeae OE48.2T, P. tensinigenes ZA5.3T, P. crudilactis UCMA 179881T, P. helmanticensis 265 CECT 8548T, P. hamedanensis SWRI65T and P. atacamensis LMG 34516T could not. We also 266 observed C14 aliphatic lipase activity in almost all strains and uncertain results for P. 267 helmanticensis CECT 8548T. Strain FIT28T exhibited aminopeptidase (arylamidase) activity 268 for leucine and valine which has been described to be related with nitrification processes [48]. 11 269 Trypsin, acid, and alkaline phosphatase activities were also observed. Aliphatic compounds 270 activity was detected by C14 lipase, C4 esterase and C8 esterase-lipase positive reaction on API 271 Zym (Biomerieux) colorimetric test. Other positive enzymatic activities such as arginine 272 dihydrolase, urease, and naphthol-AS-BI-phosphohydrolase activities were detected too. When 273 we addressed chemical growth tolerance, we also found differences between FIT28T, P. zeae 274 OE48.2T, and P. tensinigenes ZA5.3T. Strain FIT28T tolerates fusidic acid and minocycline, 275 whereas P. zeae OE48.2T is uncertain and negative, respectively. FIT28T is unable to grow in 276 the presence of lithium chloride, guanidine HCl, and D-serine, whereas P. zeae OE48.2T and 277 P. tensinigenes ZA5.3T exhibited tolerance and uncertain result, respectively. 278 In order to determine cell fatty acids and major polar lipids composition, FIT28T cell cultures 279 were grown at 28 ºC in 200 ml of TSB medium for 24h, under constant shaking at 190 rpm. 280 Cells were recovered by centrifugation at 4000 rpm for 15 min at 4 ºC. The resulting 281 supernatant was discarded, and cell pellet was resuspended in 0.22 µm sterile-filtered cryo- 282 protectant solution (ATCC Reagent-18) containing 0.75% (w/v) trypticase soy broth, 10% 283 (w/v) sucrose, and 5% (w/v) bovine serum albumin fraction V. Two hundred or 30 mg of 284 freeze-dried cells were used for polar lipids and cellular fatty acids composition analysis, 285 respectively. Results from FIT28T were compared to biochemical features and data from 286 phylogenetic-related species which were extracted from literature [39, 40, 44, 45, 47, 49, 50]. 287 The most abundant fatty acid in strain FIT28T is an aliphatic unsaturated chain of 16 carbons 288 (C16 : 0) which represents the 29.6% of the total cellular fatty acids (Table 4). This percentage 289 was slightly lower than that reported for P. crudilactis UCMA 179881T (33.7%), P. 290 helmanticensis OHA11T (31.9%), P. atagonensis (32.8%), P. granadensis LMG 27940T 291 (31.9%) and P. iridis P42T (35.97%), very similar to that in P. baetica a390T (29.43%) and P. 292 moraviensis DSM 16007T (28.8%), and clearly higher than in P. atacamensis LMG 34516T 293 (24.98%) and P. koreensis DSM 16610T (20%) which exhibited the lower C16 : 0 content among 12 294 the compared strains (Table 4). Major polar lipids were analyzed by two-dimensional thin- 295 layer chromatography (TLC). The major polar lipid species in FIT28T is 296 phosphatidylethanolamine (Figure 3). Compounds that follow in abundance are 297 diphosphatidylglycerol and phosphatidylglycerol. Finally, only traces of aminolipids, 298 phospholipids and glycolipids were detected (Figure 3). Analysis of lipids and cellular fatty 299 acids were carried out by the Identification Service, Leibniz-Institut DSMZ - Deutsche 300 Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. 301 Description of Pseudomonas germanica strain FIT28T 302 Pseudomonas germanica (ger.ma’ni.ca L. fem. adj. germanica, German, from rhizomes of the 303 Iris germanica, the source of isolation of FIT28T) was isolated from I. germanica plant- 304 rhizomes at the Marimurtra Botanical Garden, City of Blanes, Catalonia, Spain. Cells are Gram 305 negative, catalase and cytochrome oxidase positive, and have a monotrichous flagellum, hence 306 its motile capacity. Genome size was estimated at 6.713.530 of base-pairs with GC content of 307 59.09%. Genome annotation and gene prediction has unveiled 6.022 protein coding genes, 19 308 genes coding for rRNAs, and 75 genes encoding tRNAs. ANI comparison between P. 309 germanica genome and those of the nearest related species revealed 95.23% of identity with P. 310 zeae OE48.2T, 91.50% with P. tensinigenes ZA5.3T, 91.13% with P. crudilactis UCMA 311 179881T, 88.96% with P. helmanticensis OHA11T, 88.12% with P. baetica a390T, 87.73% 312 with P. atagonensis PS14T, 87.62% with P. koreensis DSM 16610T, 87.38% with P. iridis 313 P42T, 87.21% with P. granadensis LMG27940T, 86.83% with P. atacamensis M7DT and P. 314 moraviensis BS3668T, and 86.64% with P. hamedanensis SWRI65T genomes. Digital DNA- 315 DNA hybridization values were 63.4% to P. zeae OE48.2T, 46.7% to P. tensinigenes ZA5.3T, 316 45.4% to P. crudilactis UCMA 179881T, 38.6% to P. helmanticensis OHA11T, 36.4% to P. 317 baetica a390T, 35.1% to P. koreensis DSM16610T (phylogenetic subgroup of reference of the 318 P. fluorescens group of the homonym species lineage), 35% to P. atagonensis PS14T, 34.7% 13 319 to P. iridis P42T, 34.2% to P. granadensis LMG 27940T, 33.8% to P. atacamensis M7DT, 320 33.5% to P. hamedanensis SWRI65T, and 33.3% to P. moraviensis BS3668T. 321 Strain FIT28T exhibits vigorous growth on TSA medium between 4 and 35 ºC with an optimal 322 growth temperature of 28 ºC. Colonies are white-yellow and become mucoid in 16h, and the 323 pH range of tolerance is from 5 to 11, with optimal values between 6 and 7. It tolerates a 324 concentration of sodium chloride up to 6 % (w/v). Carbon source utilization is wide and 325 includes dextrin, D-trehalose, sucrose, N-acetyl-D-glucosamine, α-D-glucose, D-ribose, D- 326 mannose, D-melibiose, D-fructose, D-fucose, inosine, D-mannitol, D-arabitol, glycerol, 327 glycyl-L-proline, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L- 328 pyroglutamic acid, L-serine, pectin, D-gluconic acid, glucuronamide, xylose, mucic acid, 329 quinic acid, D-saccharic acid, methyl pyruvate, L-lactic acid, citric acid, α-ketoglutaric acid, 330 D-malic acid, L-malic acid, bromo-succinic acid, tween 40, γ-aminobutyric acid, α- 331 hydroxybutyric acid, β-hydroxy-D,L-butyric acid, propionic acid, acetic acid, and formic acid. 332 Negative oxidation and assimilation were achieved for D-maltose, D-raffinose, β-methyl-D- 333 glucoside, N-acetyl-D-galactosamine, gelatin, N-acetyl neuraminic acid, D-sorbitol, myo- 334 inositol, D-aspartic acid, gelatin, and p-hydroxyphenyl acetic acid. Strain FIT28T exhibited 335 uncertain results for D-cellobiose, gentiobiose, D-turanose, stachyose, D-salicin, N-acetyl-β- 336 D-mannosamine, 3-methyl glucose, L-fucose, L-rhamnose, D-glucose-6-phosphate, D- 337 fructose-6-phosphate, D-serine, D-galacturonic acid, L-galactonic acid lactone, D-glucuronic 338 acid, D-lactic acid methyl ester, α-ketobutyric acid, and acetoacetic acid. FIT28T displayed the 339 following enzymatic activities: acid and alkaline phosphatase, C4 esterase and C8 esterase, C14 340 lipase, arginine dihydrolase, gelatin hydrolysis, naphthol-AS-BI-phosphohydrolase, trypsin 341 and urease activity, leucine, and valine arylamidase. Regarding the chemical growth tolerance, 342 FIT28T can grow in presence of aztreonam, troleandomycin, rifamycin SV, minocycline, 343 lincomycin, vancomycin, tetrazolium violet, tetrazolium blue, nalidixic acid, potassium 14 344 tellurite, niaproof 4, fusidic acid, and 1% sodium lactate, whereas no growth was recorded for 345 guanidine HCl and lithium chloride. Major cellular fatty acids were C16:0, C18:1 ω7c, and 346 summed future 3 (C16:1 ω7c/C15:0 iso 2-OH). Detected polar lipids according to their decreasing 347 abundance were diphosphatidylglycerol, phosphatidylglycerol, aminolipids, phospholipids and 348 glycolipids. 349 Considering the genomic, physical, and biochemical differences between P. germanica and its 350 closest relatives, we expose there is enough data to consider this isolate as a newly discovered 351 bacterial species. The type strain is FIT28T (LMG 32353T = DSM 112698T). 352 Protologue 353 16s rRNA sequence, genome assembly and SRA data of P. germanica strain FIT28T was 354 deposited in Genbank of the NCBI public database under the accession number MZ758888.1, 355 ASM1961465v1 and SRR14429882, respectively. All genomic submissions are englobed into 356 BioProject number PRJNA705867, BioSample SAMN18105273. Patric 3.6.9 genome feature 357 annotation of P. zeae OE48.2T, P. tensinigenes ZA5.3T, P. iridis P42T, P. hamedanensis 358 SWRI65T, and P. helmanticensis LMG 28168T, as well, FIT28T raw genome sequence, 16S 359 rRNA and MLSA gene sequences used for phylogeny were deposited at the Microbiology 360 Society Figshare repository (https://microbiology.figshare.com/). 361 AUTHOR STATEMENTS 362 Authors and contributors 363 Kostadin Evgeniev Atanasov (KA), David Miñana-Galbis (DMG), Teresa Altabella (TA) and 364 Albert Ferrer (AF) designed experiments. KA performed experiments and analyzed data with 365 contribution of DMG, TA, and AF. Julia Gallego (JG) harvested plant material used for 366 endophyte isolation and performed strain isolation. Annabel Serpico (AS) and Montserrat 367 Bosch (MB) designed and supervised JG work. This article was written by KA with 368 contributions of all authors. 15 369 Conflicts of interest 370 Authors declare that there are no conflicts of interest. 371 Funding information 372 This work was funded by grants RTC-2017-6431 from FEDER/Ministerio de Ciencia, 373 Innovación y Universidades-Agencia Estatal de Investigación (Spain), 2017SGR710 from the 374 Generalitat de Catalunya, and by the CERCA Programme of the Generalitat de Catalunya. We 375 also acknowledge financial support from the Spanish Ministerio de Economía y 376 Competitividad-Agencia Estatal de Investigación through the “Severo Ochoa Programme for 377 Centres of Excellence in R&D” SEV-2015- 0533 and CEX2019-000902-S. 378 Acknowledgements 379 We thank Maria Jesús Montes López from the Department of Biology, Healthcare and 380 Environmental Science of the Faculty of Pharmacy and Food Sciences of the University of 381 Barcelona, for her priceless help during strain biochemical characterization. We also thanks to 382 Leigh A. Riley for help in genome annotation and submission at the NCBI GenBank, Bethesda, 383 Maryland USA 384 16 LIST OF FIGURES AND TABLES Principal figures Figure 1: Multilocus sequence analysis (MLSA) with entire 16S rRNA, rpoB, gyrB, and rpoD gene sequences. Neighbor-joining tree with pairwise deletion and bootstrap percentages on branches from 1000 replicates. Figure 2: Uranyl acetate negative stain of Pseudomonas sp. strain FIT28T. Image acquired at 25.000 magnifications. Figure 3: Thin layer chromatography (TLC) analysis of Pseudomonas sp. strain FIT28T major polar lipids. Principal tables Table 1: Average nucleotide identity (ANI) pair-comparison matrix for Pseudomonas sp. strain FIT28T and its most closely related species. Values are expressed as percentages of identity. Table 2: Digital DNA-DNA hybridization (dDDH) values between the genomes of Pseudomonas sp. strain FIT28T and its most closely related species. Table 3: Differential biochemical phenotypes between Pseudomonas sp. strain FIT28T and its closest relative species. Species 1, strain FIT28T; 2, P. zeae OE48.2T [46]; 3, P. tensinigenes ZA5.3T [46]; 4, P. crudilactis UCMA 17988T [45], 5, P. helmanticensis OHA11T (= CECT 8548); 6, P. baetica a390T (= CECT 7720); 7, P. iridis P42T [47]; 8, P. atagonensis PS14T (= CECT 9940); 9, P. koreensis DSM 16610T [37–41, 44]; 10, P. granadensis LMG 27940T [37, 39, 42, 43]; 11, P. hamedanensis SWRI65T [46]; 12, P. atacamensis LMG 34516T [37] and 13, P. moraviensis DSM 16007T [37, 39, 40, 42, 44]. Results are summarized as positive (+), negative (-), weak or uncertain (/), and not described previously (ND). API Zym enzymatic activities were scored as strong (+++), middle (++), weak (+), and uncertain (/). Table 4: Percentages of cellular fatty acids composition of strain FIT28T and its relative’s species. Specie 1, strain FIT28T; 2, P. crudilactis UCMA 17988T [45]; 3, P. helmanticensis OHA11T [50]; 4, P. baetica a390T [40]; 5, P. atagonensis PS14T [49]; 6, P. koreensis DSM 16610T [39, 41, 43]; 7, P. granadensis LMG 27940T [39, 43]; 8, P. iridis P42T [47]; 9, P. atacamensis LMG 34516T [37] and 10, P. moraviensis DSM 16007T [39, 44]. Not detected fatty acids are indicated in (-) and traces with (TR). 17 Figure 1 100 strain FIT28T (CP071586) 95 P. zeae OE48.2T (CP077090) P. crudilactis UCMA 17988T (NZ_WXVV01000001) 54 100 P. tensinigenes ZA5.3T (CP077089) P. helmanticensis LMG 28168T (PRJNA303505) 53 P. baetica LMG 25716T (NZ_PHHE01000001) 73 P. iridis P42T (NZ_JACOPU010000001) P. atagonensis PS14T (NZ_VXCA010000019) P. koreensis DSM 16610T (NZ_JAAQYM010000010) P. granadensis LMG 27940T (NZ_LT629778) 100 99 P. hamedanensis SWRI65T (NZ_CP077091.1) 100 P. atacamensis M7D1T (NZ_SSBS01000010) 100 P. moraviensis BS3668T (NZ_LT629788) P. kribbensis 46-2T (NZ_CP029608) 100 P. glycinae MS82T (NZ_CP028826) 100 100 P. yamanorum LMG 27247T (NZ_LT629793) 61 P. brenneri BS2771T (NZ_LT629800) P. migulae BS3662T (NZ_FNTY01000002) 100 P. lini DSM 16768T (NZ_JYLB01000010) 63 P. umsongensis BS3657T (NZ_LT629767) P. izuensis IzPS43-3003T (NZ_WTFT01000001) 100 P. moorei BS3775T (NZ_FNKJ01000003) 86 78 91 P. mohnii DSM 18327T (NZ_FNRV01000002) P. reinekei BS3776T (NZ_LT629709) 97 P. vancouverensis Dha-51T (NZ_RRZK01000005) 88 P. laurylsulfativorans AP3 22T (NZ_MUJK01000010) 100 99 P. jessenii BS3660T (NZ_FNTC01000002) 100 P. laurylsulfatiphila AP3_16T (NZ_NIRS01000001) P. batumici UCM B-321T (NZ_JXDG01000001) 59 90 P. asplenii ATCC 23835T (NZ_LT629777) 87 P. agarici LMG 2112T (NZ_FOAR01000077) P. lutea DSM 17257T (JRMB01000001) T P. qingdaonensis JJ3 (NZ_PHTD01000001) 84 P. laurentiana JCM 32154T (JAAHBT010000001) P. sichuanensis WCHPs060039T (NZ_QKVM01000001) P. taiwanensis DSM 21245T (NZ_KE384450) 100 P. plecoglossicida NBRC 103162T (NZ_BBIV01000097) 100 100 P. monteilii NBRC 103158T (NZ_BBIS01000132) 100 P. asiatica RYU5T (NZ_BLJF01000001) 100 P. inefficax JV551A3T (NZ_OPYN01000001) P. aeruginosa DSM 50071T (NZ_CP012001) 0.01 18 Figure 2 19 Figure 3 20 Table 1 ANI Pseudomonas sp. FIT28T 100 95.23 91.50 91.13 88.96 88.12 87.73 87.62 87.38 87.21 86.83 86.83 86.64 P. zeae OE48.2T 95.23 100 91.38 91.27 89.05 88.19 88.06 87.73 87.43 87.36 86.97 86.90 86.77 P. tensinigenes ZA5.3T 91.50 91.38 100 93.48 89.05 88.24 88.11 87.63 87.65 87.41 86.99 86.72 86.83 P. crudilactis UCMA 17988T 91.13 91.27 93.48 100 89.07 88.07 87.98 87.54 87.59 87.21 86.82 86.64 86.68 P. helmanticensis OHA11T 88.96 89.05 89.05 89.07 100 87.86 87.82 87.41 87.41 86.93 86.86 86.63 86.79 P. baetica a390T 88.12 88.19 88.24 88.07 87.86 100 87.81 87.73 88.35 86.71 86.56 86.36 86.87 P. atagonensis PS14T 87.73 88.06 88.11 87.98 87.82 87.81 100 87.60 87.47 86.64 86.62 86.42 86.51 P. koreensis DSM 16610T 87.62 87.73 87.63 87.54 87.41 87.73 87.60 100 87.16 86.69 86.55 86.49 86.44 P. iridis P42T 87.38 87.43 87.65 87.59 87.41 88.35 87.47 87.16 100 86.19 86.22 86.05 86.02 P. granadensis LMG 27940T 87.21 87.36 87.41 87.21 86.93 86.71 86.64 86.69 86.19 100.00 87.20 87.06 86.94 P. atacamensis M7DT 86.83 86.97 86.99 86.82 86.86 86.56 86.62 86.55 86.22 87.20 100 91.79 88.04 P. moraviensis BS3668T 86.83 86.90 86.72 86.64 86.63 86.36 86.42 86.49 86.05 87.06 91.79 100 88.15 P. hamedanensis SWRI65T 86.64 86.77 86.83 86.68 86.79 86.87 86.51 86.44 86.02 86.94 88.04 88.15 100 LMG 27940T DSM 16610T SWRI65T BS3668T OHA11T ZA 5.3T FIT28T UCMA 17988T PS14T M7DT a390T 48.2T P42T 21 Table 2 Species Strain dDDH to strain FIT28T P. zeae OE48.2T 63.4% [60.5 – 66.2%] P. tensinigenes ZA5.3T 46.7% [44.2 – 49.3%] P. crudilactis UCMA 17988T 45.4% [42.8 – 48.0%] P. helmanticensis OHA11T 38.6% [36.1 – 41.1%] P. baetica a390T 36.4% [34.0 – 38.9%] P. koreensis DSM 16610T 35.1% [32.6 – 37.6%] P. atagonensis PS14T 35.0% [32.5 – 37.5%] P. iridis P42T 34.7% [32.3 – 37.2%] P. granadensis LMG 27940T 34.2% [31.7 – 36.7%] P. atacamensis M7DT 33.8% [31.3 – 36.3%] P. hamedanensis SWRI65T 33.5% [31.1 – 36.0%] P. moraviensis BS3668T 33.3% [30.9 – 35.8%] 22 Table 3 Characteristics 1 2 3 4 5 6 7 8 9 10 11 12 13 Fluorescence on King's B media + ND ND - + + + + + + ND + + Growth at 37 ◦C - ND ND - - - + - + + ND + + Growth on 4% NaCl + / - + + + + + + + + ND + Growth on 6% NaCl + ND ND - - - + - + - ND - + Oxidation of: Glucose + + + + + + + + + + + + - Melibiose + / + + + + - - - + / ND - D-Arabitol + - / + + + + + + + / ND + D-Fructose + / - + + + + + + + + ND + D-Galactose + + + + + + ND + + + + - + D-Galacturonic acid / / / + / / + / - - / ND - D-Gluconic acid + / / + + + + + + ND + ND + D-Glucuronic acid / / + - + / + / + - / ND - D-Lactic acid methyl ester / - - + / + - / + ND - ND + D-Mannitol + - / + + + + + + + / ND + D-Mannose + + + + + + + + + + + ND + D-Saccharic acid + / + + + + + + + + + ND + D-Trehalose - - - - - + + - - + - + + Formic acid + - - + + + - + + - / ND - Glucuronamide + + + + + + + + + + + ND + Inosine + / / + + + + + + + + ND + 23 L-Alanine + / + + + + + + + ND + ND + L-Galactonic acid lactone / + + + / + + / + + / ND + L-Pyroglutamic acid + - + + + + + + + + + ND + Pectin + - - + / / - / ND ND - ND + p-Hydroxyphenylacetic acid - - - - - - - - - - - ND - Propionic acid + - / - + + + + + + / ND + Sucrose + - - - - - - / + + - ND ND α-Ketobutyric acid / - - - + / - / + / / ND + α-Ketoglutaric acid + + + - + + + + + + + ND + γ-Hydroxybutyric acid + - - + / / - / - ND - ND - Acetic acid + + + - + + + + + / + ND + Enzymatic activities Catalase + + + + + + + + + + + + + Cytochrome oxidase + + + + + + + + + + + + + Gelatin hydrolisis + - - - - + + + + + - - + 24 Table 4 Fatty acid 1 2 3 4 5 6 7 8 9 10 C10 : 0 3-OH 3.09 4.0 2.4 3.44 3.2 4.2 3.2 4.16 TR 2.6 C12 : 0 2-OH 3.3 5.2 4.9 5.54 5.3 6.2 4.7 5.36 10.27 4.9 C12 : 0 3-OH 3.61 4.2 2.9 3.23 4.5 5.1 2.5 4.28 TR 4.1 C12 : 0 3.26 1.4 2 1.68 1.6 5 1.5 1.7 TR 2.1 C16 : 0 29.56 33.7 31.9 29.43 32.8 20 31.9 35.97 24.98 28.8 C17 : 0 cyclo 3.06 2.8 5.1 3.15 11.5 1.5 6.9 7.48 TR 2.4 C18 : 1 ω7c 17.15 - 15.6 8.5 ND 13.4 12.4 - 9.53 17.3 C18 : 0 1.55 - 0.8 0.34 TR 1.7 - TR ND 0.5 Summed feature 3 (C16 : 1 ω7c/C15 : 0 iso 2-OH) 33.9 36.2 32.9 42.4 27.2 41.9 35.6 29.86 28.67 34.2 25 345 REFERENCES 346 1. 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