This is the accepted version of the journal article: Atanasov, K.E.; Galbis, D.M.; Cornadó, D.; [et al.]. «Pseudomonas fitomaticsae sp. nov., isolated at Marimurtra Botanical Garden in Blanes, Catalonia, Spain». International Journal of Systematic and Evolutionary Microbiology, Vol. 72 Núm. 10 (2022), p. 5557. DOI 10.1099/IJSEM.0.005557 This version is available at https://ddd.uab.cat/record/273536 under the terms of the license 1 Pseudomonas fitomaticsae sp. nov., isolated at Marimurtra Botanical Garden in Blanes, 2 Catalonia-Spain 3 4 Kostadin Evgeniev Atanasov1,2, David Miñana Galbis3, Deborah Cornadó4, Annabel Serpico4, 5 Guiomar Sánchez4, Montserrat Bosch4, Albert Ferrer1,5 and Teresa Altabella1,2 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, Universitat de Barcelona, Spain. 10 3 Department of Biology, Healthcare and the Environment, Microbiology Section, Faculty of 11 Pharmacy and Food Sciences, Universitat de 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 Universitat de Barcelona, Spain. 16 Corresponding author: Kostadin E. Atanasov 17 The email address for the corresponding author: evgenievatanassov@ub.edu 18 Keywords: Pseudomonas fitomaticsae FIT81T, multilocus sequence analysis, genome-to- 19 genome comparison, genome assembly. 20 Repositories: 21 Bio Project: PRJNA705867; Bio Sample: SAMN19241968; SRR14663887; Assembly: 22 CP075567; 16s rRNA sequence ID: MZ773500.1. 23 1 24 ABSTRACT 25 In the framework of the research project called FITOMATICS, we have isolated and 26 characterized a bacterial plant-endophyte from the rhizomes of Iris germanica, hereafter 27 referred to as strain FIT81T. The bacterium is Gram negative, rod-shaped with lophotrichous 28 flagella, and catalase and oxidase positive. The optimal growth temperature of strain FIT81T is 29 28ºC, although it can grow within a temperature range of 4ºC to 32ºC. The pH growth tolerance 30 ranges between 5 and 10, and it tolerates 4% (w/v) NaCl concentration. A 16S rRNA 31 phylogenetic analysis positioned strain FIT81T within the genus Pseudomonas, and multilocus 32 sequence analysis (MLSA) revealed that P. gozinkensis IzPS32dT, P. glycinae MS586T, P. 33 allokribbensis IzPS23T, 'P. kribbensis' 46-2, and P. koreensis PS9-14T are the top five most 34 closely related species, which were selected for further genome-to-genome comparisons, as 35 well as for physiological and chemotaxonomical characterization. The genome size of strain 36 FIT81T is 6,492,796 base-pairs in length, with 60.6% of GC content. Average nucleotide 37 identity (ANI) and digital DNA-DNA hybridization (dDDH) yielded values of 93.6% and 38 56.1%, respectively, when FIT81T genome was compared to that of the closest type-species P. 39 gozinkensis IzPS32dT. Taken together, the obtained genomic, physiologic, and 40 chemotaxonomic data indicate that strain FIT81T is different from its closest relative species, 41 which lead us to suggest that it is a novel species to be included in the list of type-strains with 42 the name Pseudomonas fitomaticsae FIT81T (=CECT 30374T =DSM 112699T). 43 2 44 INTRODUCTION 45 Land plants are soil-dependent organisms that have the capacity to interact with a plethora of 46 multikingdom microorganisms. Among these are the so-called bacterial endophytes, which can 47 enter and reside within the plant tissues without causing disease symptoms but, on the contrary, 48 play a crucial role in promoting plant growth and defense by stimulating nutrient mobilization, 49 synthesis of hormones and nutrients, and protection against biotic and abiotic stress [1, 2]. In 50 addition to their recognized potential as plant-growth promoting organisms, endophytic 51 bacteria have gained special interest because of their capacity to produce a broad range of 52 bioactive metabolites, such as bactericides, antibiotics, fungicides, antivirals, and many others 53 [3, 4]. 54 One of the most frequently genus of endophytic bacterial is Pseudomonas, which is a 55 ubiquitous Pseudomonadota, rod-shaped, aerobic, Gram negative and non-spore forming with 56 high capacity to utilize diverse compounds as carbon source, colonize a broad range of 57 eukaryotic host, and produce a wide array of specialized metabolites [5]. So far, a total of 299 58 Pseudomonas species with validly published and correct names are described for this genus 59 (https://lpsn.dsmz.de/genus/pseudomonas). 60 Pseudomonas species classification was first performed by using traditional phenotypical 61 characterization methods but, more recently, molecular techniques have emerged as key 62 analytical tools to aim species delineation. At present, multilocus sequence analysis (MLSA) 63 technique is used to study phylogenetic relationships among species. For instance, by using 64 MLSA, Peix et al. [6] suggested the division of Pseudomonas in three lineages: P. fluorescens, 65 P. aeruginosa and P. pertucinogena, from which P. fluorescens was also classified in seven 66 different groups. On the other hand, Hesse et al. [7] compared the genomes of type and none- 67 type strains, as well as of subspecies, and suggested the division of Pseudomonas in thirteen 68 groups of species, being P. fluorescens the group containing the highest number of species, 3 69 which was then divided in ten subgroups. More recently, Rudra and Gupta [8] used 118 70 concatenated conserved protein codifying genes from 255 genomes and showed a distinct 71 lineage for some halotolerant Pseudomonas species which were grouped in a Pertucinogena 72 clade. In further analysis, the authors identified 24 conserved signature indels in diverse cellular 73 functions proteins which supported the reclassification of P. pertucinogena to the new 74 proposed genus Halopseudomonas were the type species is Halopseudomonas pertucinogena. 75 In this work, we describe the characterization of a new Pseudomonas species candidate isolated 76 from the rhizomes of Iris germanica, Pseudomonas fitomaticsae, in the framework of a 77 research project (FITOMATICS) focused on the isolation and characterization of plant- 78 endophytic microorganisms with the potential to produce secondary specialized metabolites. 79 ISOLATION AND ECOLOGY 80 For bacteria isolation, rhizomes of Iris germanica from the Marimurtra Botanical Garden of 81 Blanes in Catalonia, Spain (41.67666º N; 2.80194 E) were used. Plants were examined for 82 pests, lesions or any disease symptoms, and only healthy plants were selected. Rhizomes were 83 collected in situ, maintaining plant integrity, and soil was detached manually from the 84 rhizomes. Samples were washed with sterile distilled water and phosphate buffered saline 85 solution (PBS) at pH 7.4, and surface sterilized essentially as reported by Kumar et al. [9]. The 86 rhizomes were first soaked in 70% ethanol for 5 min and then in 2% sodium hypochlorite 87 solution for 10 min, followed by 45 min in 70% ethanol. Finally, they were thoroughly washed 88 with sterile distilled water to remove any remaining ethanol. Endophytic bacteria were released 89 from the inner plant tissue by homogenizing the rhizomes in PBS. Then a two-fold serial 90 dilution of the crude-extract was prepared, and 50 µl of each sample were spread on Tryptic 91 Soy Agar medium (TSA, Sigma-Aldrich, St Louis, USA) plates, which were incubated at 30ºC 92 for up to two weeks. As soon as colonies appeared, they were individually transferred to 3 ml 93 of Tryptic Soy Broth (TSB, Becton Dickinson, Franklin Lakes, USA) medium and cultured 4 94 over-night at 30ºC with constant shaking at 190 rpm. After endophyte extraction, the rhizomes 95 were incubated in TSA medium at 30ºC for up to three weeks, and no bacterial growth was 96 observed, thus demonstrating the effectiveness of the sterilization procedure. For long-term 97 strain preservation, 800 µl of an over-night culture were mixed with 200 µl of 80% sterile 98 glycerol and stored at -80ºC. 99 PHYLOGENETIC ANALYSIS 100 For DNA extraction, 2 ml of strain FIT81T over-night TSB culture was used. Cells were 101 recovered by centrifugation at 11,000 g for 3 min and the genomic DNA was extracted with 102 Wizard Genomic DNA Extraction Kit according to manufacturer´s instructions (Promega, 103 Madison, USA). Thirty ng of the genomic DNA was used for the 16S rRNA gene PCR 104 amplification with the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 105 1492R (5’-GGTTACCTTGTTACGACTT-3’) [10], and AccuPrime Pfx polymerase (2.5 units) 106 (Thermo Fisher Scientific, Waltham, USA). The PCR program was as follows: DNA 107 denaturalization at 95ºC for 5 min, 25 cycles of amplification including denaturalization at 108 95ºC for 15 s, annealing at 50ºC for 30 s, 2 min extension at 72ºC, and 10 min at 72ºC for 109 complete synthesis of amplification product. The PCR product was analyzed by agarose gel 110 electrophoresis, purified using PCR clean-up columns (Macherey-Nagel GmbH & Co. KG, 111 Düren, Germany), and SANGER-sequenced using primers 27F or 1492R. The resulting partial 112 sequences were assembled in a unique consensus sequence with the DNA Dragon software 113 (SequentiX, Klein Raden, Germany), which was analyzed by nucleotide BLAST (blastn) 114 alignment on the NCBI database (https://www.ncbi.nlm.nih.gov/) under the parameters of 115 standard database, uncultured/environmental sample sequence exclusion, and search limited to 116 type material. Blast results positioned strain FIT81T within the genus Pseudomonas with 100% 117 of sequence similarity to 'P. kribbensis' 46-2, P. glycinae MS586T and P. saponiphila DSM 118 9751T. Next, the entire 16S rRNA sequence was extracted from the FIT81T genome annotation, 5 119 and a species search on the EzBioCloud taxonomically united database [11] was performed. A 120 total of 33 species were selected from the EzTaxon and 13 from the NCBI nucleotide blast. H. 121 pertucinogena (homotypic synonym of P. pertucinogena) NBRC 14163T (AB680571) was 122 used as a rooting group. The 16S rRNA sequences were aligned by the MUSCL algorithm [12] 123 and a phylogenetic tree was constructed by the Maximum Likelihood method based on the 124 Jukes-Cantor mode with MEGA7 [13, 14]. This analysis outputted an unprecise tree-branch 125 clustering of strain FIT81T, showing 99.9% of sequence similarity to P. gozinkensis IzPS32dT, 126 'P. kribbensis' 46-2, and P. glycinae MS4586T (Fig. S1; Table S1). Hence, by using 16S rRNA 127 phylogeny, it was not possible to locate the newly isolated strain in any subgroup of the P. 128 fluorescens group [7]. Thus, a MLSA was conducted to improve resolution on the strain 129 phylogeny. Protein-coding housekeeping genes rpoB, gyrB and rpoD were extracted from the 130 NCBI genome annotation, whereas for some species genomes were annotated by Patric 3.6.12 131 with the Rapid Annotation using Subsystems Technology algorithm (RAST) [15]. Sequences 132 of the 16S rRNA, rpoB, gyrB, and rpoD genes were concatenated, aligned with MUSCL 133 algorithm, and a new MLSA-based Maximum Likelihood tree constructed (Fig. 1). The overall 134 sequences similarities of the FIT81T concatenated housekeeping genes were as follows: 98.2% 135 to P. gozinkensis IzPS32dT and P. glycinae MS4586T; 97.9% to 'P. kribbensis' 46-2; 97.4% to 136 P. allokribbensis IzPS23T and 97.0% to the P. koreensis PS9-14T (Table S2). These results 137 further supported the view that strain FIT81T is closely related to P. glycinae MS4586T and P. 138 gozinkensis IzPS32dT and positioned strain FIT81T as a new candidate of the P. koreensis 139 subgroup of the P. fluorescens group with a clear clustering differentiation to other P. jessenii 140 and P. gessardii subgroups. Thus, the selection of species for the whole genome-based species 141 comparisons and the physiological and chemical characterization was based on these MLSA- 142 based phylogenetic relationships. 143 6 144 GENOME FEATURES 145 Genomic DNA from strain FIT81T was extracted as described above, treated with RNase A 146 DNase free (Panreac AppliChem, Darmstadt, Germany), and deproteinized by chloroform- 147 isoamyl alcohol treatment. The DNA was cleaned and concentrated by using Genomic DNA 148 Clean & Concentrator-10 columns (Zymo Research, Irvin, USA), and whole genome 149 sequencing was carried out by combining the Illumina Novaseq (2x150 bp) and PacBio RSII 150 platforms. The resulting sequence data were combined to generate a hybrid genome assembly 151 by using Unicycler software [16]. Genome size of FIT81T is 6,492,796 base-pair length with 152 60.6% of GC content, which agrees with genome size and GC content of Pseudomonas species 153 isolated from environmental samples [17]. Gene annotation was performed by NCBI 154 Prokaryotic Genome Annotation Pipeline (PGAP) [18–20]. A total number of 5,742 protein- 155 coding genes, 19 rRNA coding genes (7 genes coding for 5S rRNAs, 6 genes coding for 16S 156 rRNAs and 6 genes coding for 23S rRNAs), and 72 tRNAs coding genes were predicted. 157 Genome circularization was achieved by visualization of FIT81T genome with Bandage 158 software [21]. 159 Furthermore, genome-to-genome computational comparisons were conducted between FIT81T 160 and the top five related species. The average nucleotide identity (ANI) and orthologous ANI 161 (orthoANI) values obtained using the orthoANI Tool v0.93.1 [22] were, 93.6% and 94.2% to 162 the P. gozinkensis IzPS32dT; 93.6% and 94.0% to P. glycinae MS4586T; 91.0% and 91.5% to 163 P. allokribbensis IzPS23T; 90.9% and 91.3% to 'P. kribbensis' 46-2, 87.5% and 87.9% to the 164 P. koreensis PS9-14T, respectively. Usearch ANI analysis (ANIu) was performed with the ANI 165 calculator web-based tool from the EzBioCloud [23] and ANI maximal unique matches 166 (ANIm) estimated by JSpeciesWS [24]. The obtained ANIu and ANIm values were very 167 similar to those from the orthoANI analysis. On the other hand, values obtained from the 168 BLAST-based ANI (ANIb) calculated with JSpeciesWS were like the original ANI (Table 1). 7 169 Finally, we estimated the DNA-DNA hybridization (dDDH) values between species with the 170 Genome-to-Genome Distance Calculator (GGDC) from the DSMZ webtool (https://ggdc- 171 test.dsmz.de/ggdc.php#), with the BLAST+ local alignment algorithm using formula 2, which 172 consists of the sum of all identities within high-scoring segment pairs (HSPs) divided per 173 overall HSP length [25, 26]. Values from the dDDH were as follows: 56.1% [53.4 - 58.8%] to 174 P. gozinkensis IzPS32dT; 54.9% [52.1 - 57.6%] to P. glycinae MS4586T; 44.4% [41.9 - 47.0%] 175 to P. allokribbensis IzPS23T; 43.9% [41.4 - 46.5%] to 'P. kribbensis' 46-2; 34.7% [32.2 - 176 37.2%] to the P. koreensis PS9-14T. All these genome-to-genome comparison percentages 177 were below the threshold of 95-96% (ANI) and 70% (dDDH) for species boundaries 178 delineation [27, 28], indicating there is enough nucleotide identity and dDDH differences to 179 propose that strain FIT81T is a different species from its closest relatives, which is further 180 supported by the recently described methodology of bacteria and subspecies delineation 181 proposed by Meier-Kolthoff on the Type (Strain) Genome Server (TYGS; https://tygs.dsmz.de) 182 for prokaryote taxonomy [29]. FIT81T genome was uploaded on the TYGS server and a total 183 number of 14 out of 15,682 type strains available in the TYGS database passed the analysis as 184 closest type strains. The position of FIT81T in the phylogenetic tree constructed using the 185 Genome Blast Distance Phylogeny (GBDP) calculator (Fig. 2) agreed with the MLSA 186 phylogeny and the genome-to-genome comparison results. 187 GENOME MINING AND IDENTIFICATION OF ENDOPHYTIC LIFE-STYLE 188 RELATED GENES 189 Genome assemblies of strains FIT81T, P. gozinkensis IzPS32dT, and P. glycinae MS4586T were 190 analyzed by using the Antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) 6.0 191 webserver [30]. This computational analysis predicted the occurrence of 14 regions containing 192 10 putative biosynthetic gene clusters (BGCs) (Table S3). According to this, known cluster 193 similarities were found for the cyclic lipopeptide lokisin (71%; Pseudomonas sp. [31]), the 8 194 oxidative stress protectant aryl polyene (APE) Vf or methyl (2E,4E,6E,8E,10E,12E)-13-(4- 195 hydroxy-3,5-dimethylphenyl)trideca-2,4,6,8,10,12-hexaenoate (40%; Aliivibrio fischeri 196 ES114 [32]), the detoxing compound rimosamide (20%; Streptomyces rimosus subsp. rimosus 197 ATCC 10970 [33]), the chromophoric siderophore pyoverdine (21%, 20%, and 11%; 198 Pseudomonas protegens Pf-5[34, 35] ), the bioactive cyclic lipopeptide fengycin (13%; 199 Bacillus velezensis FZB42 [36, 37]), and the antibiotic lankacidin C (13%; [38–42]). 200 Interestingly a similar cluster prediction was obtained by antiSMASH among the three bacterial 201 species. Nevertheless, differences were found regarding the BGC for the nonribosomal 202 bioactive cyclic lipopeptide, bacillomycin D, which was predicted into the genome of P. 203 glycinae MS4586T (20%; B. velezensis FZB42 [43]), but not in FIT81T nor in P. gozinkensis 204 IzPS32dT genomes. In addition, subtle differences exist concerning the BGC for the antifungal 205 compound fragin, which was detected in P. gozinkensis IzPS32dT and P. glycinae MS4586T 206 (37%; Burkholderia cenocepacia H111 [44]), but not in FIT81T (Table S3). Genomic regions 207 containing BGCs were also annotated by using the Minimum Information about a Biosynthetic 208 Gene cluster (MIBiG [45]) repository, and the top three similarity score-based compounds 209 annotated (Table S3). To summarize, the genome mining analysis suggested a high 210 conservation of the lokisin BGC and variation some among species in the case of the BGCs for 211 bacillomycin D and fragin. The usefulness of those clusters as species and subspecies 212 identification markers could be further investigated. 213 During last decade, several genes have been suggested to serve as markers for the identification 214 of bacterial endophytes [46–51]. Thus, besides the genome mining, we performed a 215 computational search to identify endophytic-capacity life-style genes. This analysis revealed 216 that several endophyte gene markers potentially involved in phosphate solubilization, hormone 217 synthesis and response, metabolite transport, secretion and delivery systems, cell wall 218 attachment, plant polymer/modification, transcriptional regulators, siderophores, and 9 219 detoxification are present in the genomes of FIT81T, P. gozinkensis IzPS32dT, and P. glycinae 220 MS586T (Table S4). Interestingly, a detoxification-related gene coding for acetaldehyde 221 dehydrogenase was found only in the genome of FIT81T. These results together with the way 222 in which FIT81T was isolated would confirm the endophytic nature of FIT81T. 223 PHYSIOLOGY AND CHEMOTAXONOMY 224 Growth of strain FIT81T is vigorous and forms white-mucoid colonies. The bacterium can grow 225 within a temperature range from 4ºC to 32ºC in TSA medium, being 28ºC the optimal growth 226 temperature. The pH growth tolerance in TSB medium was recorded within the range of pH5 227 to pH10, even though FIT81T can resist alkaline conditions up to pH 12, at this pH growth is 228 seriously compromised. Limited growth capacity was observed at pH 4, and no growth was 229 observed at lower pH values after three weeks. Strain FIT81T is a Gram-negative bacterium. 230 Uranyl acetate negative stain and transmission electron microscope (TEM) Bioscan Gatan, 231 JEM-1010 (JEOL) acquired image were used to visualize its lophotrichous flagella and to 232 measure the average cell size, which is 2±0.3 µm length and 0.8±0.2 µm width (N=10) (Fig. 233 3). Fluorescence production was observed when FIT81T was cultured on solid King’s B 234 medium (Duchefa Biochemie, Haarlem, TheNetherlands) at 28ºC and irradiated by 365 nm UV 235 light [52]. 236 Polar lipids and fatty acids methyl esters (FAME) were determined by the Identification 237 Service, Leibniz - Institute DSMZ – Deutsche Sammlung von Mikroorganismen und 238 Zelkulturen GmbH (Braunschweig, Germany) from an over-night culture of FIT81T in TSB 239 medium incubated at 28ºC with shaking at 190 rpm. Cells were pelleted by centrifugation at 240 4,000 g for 10 min at 4ºC, mixed with cryoprotectant ATCC Reagent-18 241 (https://www.atcc.org/resources/culture-guides/bacteriology-culture-guide), and freeze-dried 242 until further analysis. Cellular fatty acids from 30 mg of cells biomass were analyzed after 243 conversion into fatty acid methyl esters (FAMEs) by saponification, methylation and extraction 10 244 using minor modifications of the methods described by Miller [53] and Kuykendall et al. [54]. 245 The FAME mixtures were then separated with gas chromatography and detected by a flame 246 ionization detector using Sherlock Microbial Identification System (MIS) (MIDI, Microbial 247 ID, Newark, DE 19711 U.S.A.). Peaks were automatically integrated, and fatty acid were 248 identified and quantified by the MIS Standard Software (Microbial ID). Summed feature 3 was 249 resolved by a GC-MS run on a Agilent GC-MS 7000D using an Agilent HP-5 ms UI 30 m x 250 250 μm x 0.25μm column, with a helium flow of 1.2 ml, with an injection of 1 μl with split 251 ratio of 7.5:1. The oven program was as follows: initial temperature 170ºC, ramp 3ºC/min to 252 200°C, ramp 5°C/min to 270°C, ramp 120°C/min to 300°C and hold for 2 min. The inlet 253 temperature was set to 170°C and then linearly increased with 200°C/min up to 350°C and hold 254 for 5 min. The mass spectrometry parameters were set to aux temperature 230°C, source 255 temperature 230°C, and electron impact ionization at 70 eV with mass range of m/z 40-600 or 256 40-800, respectively. Peaks were identified based on retention time and mass spectra. The 257 position of single and double bounds was confirmed by derivatization to the corresponding 258 dimethyl disulfide adducts [55]. Branched-chain fatty acid positions, cyclo-positions and 259 multiple double bounds were determined by conversion to their 3-pyridylcarbinol and/or 4,4- 260 dimethyloxazoline (DMOX) derivatives [56–58]. 261 FIT81T showed a Pseudomonas species typical profile C10:0 3-OH, C12:0, and C12:0 3-OH fatty 262 acids, which further supported its affiliation to genus Pseudomonas [59]. In addition, the major 263 FAME was the unsaturated sixteen carbon aliphatic chain (C16:0) with an average value of 30.6, 264 which is slightly lower than those found in P. gozinkensis IzPS32dT, P. allokribbensis IzPS23T 265 [20], 'P. kribbensis' 46-2 [60], and P. koreensis PS9-14T, but higher than those described for P. 266 glycinae MS586T [61]. Other fatty acids detected as FAMEs, were C18:1 ω7c (14.4); C12:0 3- 267 OH (4.4); C10:0 3-OH (4.1); C12:0 2-OH (3.9); C17:0 cyclo (3.8); C12:0 (3.7); C18:0 (1.48), and 268 traces of C10:0 , C12:1 3-OH, and C14:0. Moreover, the summed feature 3 of C16:1 ω7c/C15:0 iso 2- 11 269 OH was clearly higher in FIT81T than the levels found in P. gozinkensis IzPS32dT [20], P. 270 glycinae MS586T [61], P. allokribbensis IzPS23T [20], and 'P. kribbensis' 46-2 [60] (Table 2). 271 Polar lipids (PL) were extracted from 200 mg of freeze-dried cell material using a 272 choroform:methanol:0.3% aqueous NaCl mixture and recovered into the chloroform phase 273 [62]. PL were then separated by two-dimensional silica gel thin layer chromatography (TLC) 274 by using chloroform:methanol:water and chloroform:methanol:acetic acid:water as mobile 275 phase for first and second direction, respectively. Total lipid material was detected by using 276 molybdatophosphoric, acid and specific functional groups were identified using spray reagents 277 specific for defined functional groups [63]. The detected major compounds species were 278 phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), 279 glycolipid (GL), phospholipid (PL), and aminolipid (AL) (Fig. 4). 280 Carbon source utilization and chemical sensitivity was assessed with the miniaturized 281 microplate assay of Biolog’s GenIII (Biolog, Inc., Hayward, USA) following manufacturer´s 282 instructions. The main differences between FIT81T, P. gozinkensis IzPS32dT, and P. glycinae 283 MS586T were found in the utilization of D-arabitol, D-serine, D-fructose-6-phosphate, D- 284 galactose, pectin, D-fucose, inosine, D-galacturonic acid, L-galactonic acid lactone, 285 glucuronamide, D-malic acid, bromo-succinic acid, α-keto-butyric acid, acetoacetic acid, and 286 tween 40 (Table 3). FIT81T exhibited chemical sensitivity to D-serine, guanidine HCl, lithium 287 chloride, sodium butyrate, and sodium bromate, but it was tolerant to sodium lactate, fusidic 288 acid, troleandomycin, rifamycin SV, minocycline, lincomycin, niaproof 4, vancomycin, 289 tetrazolium violet, tetrazolium blue, nalidixic acid, potassium tellurite, and the monobactam 290 aztreonam. FIT81T shares high chemical compound tolerance with P. gozinkensis IzPS32dT 291 [23], but differences were found in the case of fusidic acid and minocycline. Enzyme activities 292 of strain FIT81T were analyzed on API 20 NE and API Zym strips (Biomerieux, Marcy-l'Étoile, 293 France). The results obtained revealed that FIT81T exhibits arginine dihydrolase, esculin 12 294 hydrolysis (β-glucosidase), and gelatin hydrolysis (protease) activities. It can also grow with 295 N-acetyl-glucosamine while P. gozinkensis IzPS32dT cannot. FIT81T and P. gozinkensis 296 IzPS32dT have both C4 and C8 esterase lipase activity. Moreover, FIT81T exhibited alkaline 297 phosphatase, C14 lipase, trypsin, naphthol-AS-BI-phosphohydrolase, and acid phosphatase 298 activities, which have not been described in P. gozinkensis IzPS32dT. It is worth noting that 299 acid phosphatase activity has been reported as a mechanism of phosphate solubilization from 300 organic compounds, which further supports our claim that FIT81T is an endophytic bacterium. 301 Finally, the above biochemical comparisons among the five closest relative species to FIT81T, 302 as well as additional biochemical traits screened in API 20E and API 50CH, are summarized 303 in more detail in Table 3. 304 In conclusion, the results from de novo genome sequencing, and the pangenome computational 305 comparative analyses based on the top five phylogenetically-related species to strain FIT81T, 306 together with a thorough characterization of the bacterium chemo-physiological traits, leads to 307 the claim that strain FIT81T represents a novel Pseudomonas species. 308 DESCRIPTION OF PSEUDOMONAS FITOMATICSAE SP. NOV. 309 Pseudomonas fitomaticsae (fi.to.ma'tics.ae. N.L. gen. n. fitomaticsae, referring to the 310 FITOMATICS research project), strain FIT81T, isolated from the rhizomes of Iris germanica 311 accessed at the Marimurtra Botanical Garden, from Blanes, Catalonia, Spain (41.67666º N; 312 2.80194 E). 313 The bacterium is Gram negative, rod-shaped with lophotrichous flagellum, and its average cell 314 size is 2±0.3 µm length and 0.8±0.2 µm width. Its optimal growth temperature is 28ºC, forming 315 white mucoid colonies in TSA medium, although it is also able to grow in a range of 316 temperatures comprised between 4ºC and 32ºC. Growth was observed at pH values ranging 317 from 5 to 10, with higher tolerance to alkaline pH than acidic one. The genome size is 6,492,796 318 base-pairs, with 60.6% GC content, and is predicted to harbor 5,742 protein-coding genes and 13 319 19 rRNA-coding genes. Among the latter, 7 genes are predicted to encode the 5S rRNA, 6 320 genes the 16S rRNA, and 6 genes the 23S rRNA. The type-strain of P. fitomaticsae FIT81T 321 was deposited at international recognized cell-type culture collection (FIT81T =CECT 30374T 322 =DSM 112699T) and is available for further biochemical, physiological, taxonomic, and 323 genetic studies. Sequence Read Archive (SRA) was deposited at the National Center for 324 Biotechnology Information (NCBI) database under the identification codes SRR14663887, 325 PRJNA705867 (Bio Project), and SAMN19241968 (Bio Sample). 16S rRNA sequence was 326 deposited in GenBank under the identification code MZ773500, and genome assembly was 327 assigned the code CP075567. 328 Authors and contributors 329 Kostadin E. Atanasov (KA) designed experiments with the contribution of David Miñana- 330 Galbis (DMG), Teresa Altabella (TA) and Albert Ferrer (AF). KA conducted experiments and 331 analyzed data with contribution of DMG, TA, and AF. Deborah Cornadó (DC) isolated strain 332 FIT81T. Annabel Serpico (AS) participated in strain isolation and culturing. Guiomar Sánchez 333 (GS) and Montserrat Bosch (MB) designed and supervised DC and AS work. This article was 334 written by KA with contributions of all authors. 335 Conflicts of interest 336 Authors honestly declare that there are no economical, ethical, or publishing conflicts of 337 interest. 338 Funding information 339 This work was funded by grants RTC-2017-6431 from FEDER/Ministerio de Ciencia, 340 Innovación y Universidades-Agencia Estatal de Investigación (Spain), 2017SGR710 from the 341 Generalitat de Catalunya, and by the CERCA Programme of the Generalitat de Catalunya. We 342 also acknowledge financial support from the Spanish Ministerio de Economía y 14 343 Competitividad-Agencia Estatal de Investigación through the “Severo Ochoa Programme for 344 Centres of Excellence in R&D” SEV-2015- 0533 and CEX2019-000902-S. 345 Acknowledgements 346 We thank to Leigh A. Riley for help in genome annotation and submission at the NCBI 347 GenBank, Bethesda, Maryland USA. 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Pseudomonas koreensis sp. 523 nov., Pseudomonas umsongensis sp. nov. and Pseudomonas jinjuensis sp. nov., novel species 524 from farm soils in Korea. Int J Syst Evol Microbiol 2003;53:21-27. 23 525 Figure legends 526 Fig. 1. Multilocus sequence analysis (MLSA) phylogenetic tree based on the 16S rRNA, rpoB, 527 gyrB, and rpoD concatenated sequences with an average length of 9,791 nucleotides showing 528 the position of strain FIT81T among related species. Sequences were aligned by MUSCL 529 algorithm, and the phylogenetic tree was constructed with MEGA 7 software by using 530 Maximum Likelihood statistical method, Jukes-Cantor substitution method, and pairwise 531 deletion for the missing data. Bootstrap values >50% based on 1000 replications are indicated 532 on branches. Bar, 1 nt substitution per 100 nt. 533 Fig. 2. Strain FIT81T Genome Blast Distance Phylogeny (GBDP) phylogenetic tree based on 534 genome data retrieved from the TYGS server. Branch lengths were calculated with formula D5 535 and the algorithm of greedy with trimming. Pseudo-bootstrap values >60% based on 100 536 replications are indicated on branches. Bar, 1 nt substitution per 100 nt. 537 Fig. 3. Strain FIT81T uranyl acetate negative stain and image captured by transmission electron 538 microscopy (TEM). 539 Fig. 4. Two-dimensional thin-layer chromatography (TLC) of the polar lipids fraction of strain 540 FIT81T. The position of phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), 541 phosphatidylglycerol (PG), glycolipid (GL), phospholipid (PL), and aminolipid (AL) is 542 indicated by arrows. 24 543 Table 1. Genome-to-genome computational relatedness of the top-five strain FIT81T closest related species. original ortho ANIb and ANIm and Genome GC ANIu dDDH and Species ANI ANI [% aligned [% aligned size (bp) (%) (%) [% model CI] (%) (%) nucleotides] nucleotides] strain FIT81T 6.492.796 60.6 - - - - - - P. gozinkensis IzPS32dT 6.563.527 60.3 93.6 94.3 94.0 93.7 [83.2] 94.5 [84.8] 56.1% [53.4 - 58.8%] P. glycinae MS586T 6.396.728 60.5 93.6 94.0 93.8 93.2 [82.5] 94.4 [82.3] 54.9% [52.1 - 57.6%] P. allokribbensis IzPS23T 6.565.027 60.3 91.0 91.5 91.5 90.9 [82.5] 91.9 [82.6] 44.4% [41.9 - 47.0%] 'P. kribbensis' 46-2 6.324.282 60.5 90.9 91.3 91.3 90.7 [81.5] 91.8 [81.3] 43.9% [41.4 - 46.5%] P. koreensis PS9-14T 6.123.913 60.5 87.5 87.9 87.5 86.9 [72.3] 89.0 [71.7] 34.7% [32.2 - 37.2%] 544 25 545 Table 2. Main cellular fatty acids detected as FAMEs in strain FIT81T and the closest related 546 species. Strains: 1, strain FIT81T; 2, P. gozinkensis IzPS32dT [20]; 3, P. glycinae MS586T [61]; 547 4, P. allokribbensis IzPS23T [20]; 5, 'P. kribbensis' 46-2 [60], and 6, P. koreensis Ps9-14T [64]. 548 Data for FIT81T were obtained in this study whereas for other species were extracted from Fatty acid 1 2 3 4 5 6 C10 : 0 TR ND TR ND ND ND C10 : 0 3-OH 4.1 4.2 6.6 4.1 5.4 2.2 C12 : 0 2-OH 3.9 4.9 5.5 5.1 6.8 5.0 C12 : 0 3-OH 4.4 5.0 6.7 5.4 7.5 4.0 C12 : 1 3-OH TR ND ND ND ND ND C12 : 0 3.7 2 2.9 1.8 ND TR C14 : 0 TR TR TR TR 1.2 ND C16 : 0 30.6 32.4 22.9 31.9 33.4 33 C17 : 0 cyclo 3.8 17.7 10.3 16.1 15.1 2.0 C18 : 1 ω7c 14.4 ND ND ND ND ND C18 : 0 1.5 TR TR TR 1.6 TR Summed feature 3 (C16 : 1 ω7c/C15 : 0 iso 2-OH) 30.3 18.3 23.6 19.8 16.8 37 549 literature. 550 26 551 Table 3. Biochemical features of strain FIT81T and its related species. 552 Strains: 1, strain FIT81T; 2, P. gozinkensis IzPS32dT; 3, P. glycinae MS586T; 4, P. 553 allokribbensis IzPS23T; 5, 'P. kribbensis' 46-2, and 6, P. koreensis Ps9-14T. Data for FIT81T, 554 P. glycinae MS586T (= LMG 30275T), and 'P. kribbensis' 46-2 (= DSM 100278T) were 555 obtained in this study whereas those for P. gozinkensis IzPS32dT, P. allokribbensis IzPS23T, 556 and P. koreensis Ps9-14T were extracted from literature [20, 64]. Symbols legend are positive 557 (+), negative (-), weak or uncertain (w), variability in literature (-/+), and not determined (ND). Characteristics 1 2 3 4 5 6 Fluorescence (fluorescein pigment) + + + + - + Growth at 37ºC - - - - - -/+ Growth on 4% NaCl + + + + + + Carbon source utilization: D-Arabitol + - + - + + D-Aspartic acid - - - - w + D-Serine - + + - w ND α-D-Glucose + + + + + + D-Fructose-6-phosphate w - w - + ND N-Acetyl-D-glucosamine + - + - + + D-Galactose + w + w + + Pectin w - w - w ND Citrate + + + + + + D-Fucose w - w - + ND D-Mannose + + + + + + Inosine + - + - + + D-Galacturonic acid w - w - + -/+ L-Arabinose + + + + + + Glycerol + + + ND + + L-Galactonic acid lactone w - w - + + D-Glucuronic acid w - w - + -/+ Glucuronamide + - + - + + D-Malic acid + - w - + ND Mucic acid + + + w + ND Bromo-succinic acid + - + - + ND α-Keto-butyric acid w - w - w -/+ Acetoacetic acid w - w - w ND Quinic acid + + + + + + 27 D-Saccharic acid + + + - + + Tween 40 + w + - + + Enzymatic activity Alkaline phosphatase + + + + + ND C4 esterase + + + + + ND C8 esterase lipase + + + + + ND C14 lipase + ND + ND + ND Leucin arylamidase w + + + w ND Valine arylamidase w + + + w ND Cystine arylamidase w ND w ND w ND Trypsin + ND + ND + ND α-Chymotrypsin w ND w ND w ND Acid phosphatase + + + + + ND Naphthol-AS-BI-phosphohydrolase + + + + + ND Lysine decarboxylase - ND - ND + ND Arginine dihydrolase + ND + ND + + Ornithine decarboxylase - ND - ND - ND Gelatin hydrolysis (protease) + ND + ND + + Urease + ND + ND + ND Esculin hydrolysis (β-glucosidase) + ND + ND + ND β-Galactosidase - ND - ND - ND (p-nitrophenyl-β-D-galactopyranosidase) 558 28 Pseudomonas fitomaticsae sp. nov., isolated at Marimurtra Botanical Garden in Blanes, Catalonia- Spain Kostadin Evgeniev Atanasov1,2, David Miñana Galbis3, Deborah Cornadó4, Annabel Serpico4, Guiomar Sánchez4, Montserrat Bosch4, Albert Ferrer1,5 and Teresa Altabella1,2 1 Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, Barcelona, Spain. 2 Department of Biology, Healthcare and the Environment, Plant Physiology Section, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona, Spain. 3 Department of Biology, Healthcare and the Environment, Microbiology Section, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona, Spain. 4 Applied Microbiology and Biotechnology Unit, LEITAT Technological Center, Terrassa, Spain. 5 Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona, Spain. Corresponding author: Kostadin E. Atanasov The email address for the corresponding author: evgenievatanassov@ub.edu 1 List of Supplemental Figures and Tables Supplemental Figure 1 (Fig. S1): Phylogenetic tree constructed on the 1,453 average 16S rRNA sequences length of the strain FIT81T relatives. Sequence alignment was performed by using MUSCL algorithm and tree constructed by the statistical method of Maximum Likelihood, Jukes-Cantor substitution method, and pairwise deletion of gaps or missing data. Bootstrap test of phylogeny was performed with 1000 replications and values >50% indicated on branches. Bar, 1 nt substitution per 100 nt. Supplemental Table 1 (Table S1): Species, strain designation, accession ID, and sequence identity for the 16S rRNA nucleotide sequence blast analysis of strain FIT81T and its closest relative species. Supplemental Table 2 (Table S2): Species, strain designation, assembly ID, and sequence identity for the MLSA nucleotide blast of strain FIT81T and its closest relative species. Supplemental Table 3 (Table S3): Genome mining of strain FIT81T, P. gozinkensis IzPS32dT; 3, P. glycinae MS586T. Supplemental Table 4 (Table S4): Identification of endophytic behavior genes: 1, strain FIT81T; 2, P. gozinkensis IzPS32dT; 3, P. glycinae MS586T. 2 Fig. S1 50 P. soli F-279208T (HF930598) P. entomophila L48T (AY907566) P. monteilii NBRC 103158T (BBIS01000088) P. inefficax JV551A3T (OPYN01000008) P. plecoglossicida NBRC 103162T (BBIV01000080) T 84 P. asiatica RYU5 (MH517510) P. taiwanensis BCRC 17751T (EU103629) P. parafulva NBRC 16636T (BBIU01000051) T 95 P. fulva NBRC 16637 (BBIQ01000036) P. defluvii WCHP16T (KY979145) 56 P. lutea DSM 17257T (JRMB01000004) 50 P. rhizosphaerae IH5T (AY152673) P. eucalypticola NP-1T (MN238862) P. koreensis Ps9-14T (AF468452) P. atacamensis M7D1T (SSBS01000008) P. moraviensis CCM 7280T (AY970952) strain FIT81T (MZ773500) P. gozinkensis IzPS32dT (CP062253) 'P. kribbensis' 46-2 (AF468452) P. glycinae MS586T (MG692779) 'P. batumici' UCM B-321 (JXDG01000105) P. hamedanensis SWRI65T (NZ CP077091) P. allokribbensis IzPS23T (CP062252) P. atagonensis PS14T (MN396717) P. granadensis LMG 27940T (LT629778) P. turukhanskensis IB1.1T (KP306892) P. neuropathica P155T (LR797591) P. helmanticensis OHA11T (HG940537) T 60 P. tensinigenes ZA5.3 (NZ CP077089) T P. zeae OE48.2 (NZ CP077090) P. crudilactis UCMA 17988T (NZ WXVV01000001) P. baetica a390T (FM201274) P. reinekei Mt-1T (AM293565) P. umsongensis DSM 16611T (NIWU01000003) P. vancouverensis ATCC 700688T (AJ011507) P. moorei RW10T (AM293566) P. jessenii DSM 17150T (NIWT01000013) P. mohnii DSM 18327T (FNRV01000001) P. izuensis IzPS43 3003T (MN865785) P. laurylsulfativorans AP3 22T (MF554631) P. laurylsulfatiphila AP3_16T (KY462012) P. agarici NCPPB 2289T (AKBQ01000002) P. iridis P42T (LR797554) P. mucoides P154aT (LR797589) P. mucidolens DSM 19186T (KX186963) 90 P. gessardii CFML 95-251T (AF074384) P. saponiphila DSM 9751T (FM208264) H. pertucinogena NBRC 14163T (AB680571) 0.01 3 Table S1 Species Strain Accession ID Identity Cover Length E value 'P. kribbensis' 46-2 AF468452 99.9% 95% 1459 0.0 P. gozinkensis IzPS32dT CP062253 99.9% 100% 1533 0.0 P. glycinae MS586T MG692779 99.9% 95% 1459 0.0 P. allokribbensis IzPS23T CP062252 99.8% 100% 1533 0.0 P. atacamensis M7D1T SSBS01000008 99.7% 95% 1459 0.0 P. moraviensis CCM 7280T AY970952 99.6% 95% 1459 0.0 P. granadensis LMG 27940T LT629778 99.6% 95% 1459 0.0 P. koreensis Ps9-14T AF468452 99.6% 94% 1455 0.0 P. reinekei Mt-1T AM293565 99.4% 95% 1459 0.0 P. hamedanensis SWRI65T CP077091 99.4% 100% 1542 0.0 P. vancouverensis ATCC 700688T AJ011507 99.3% 95% 1459 0.0 P. jessenii DSM 17150T NIWT01000013 99.3% 95% 1459 0.0 P. baetica a390T FM201274 99.2% 94% 1446 0.0 P. neuropathica P155T LR797591 99.2% 80% 1232 0.0 P. atagonensis PS14T MN396717 99.2% 95% 1460 0.0 P. izuensis IzPS43_3003T MN865785 99.1% 95% 1459 0.0 'P. batumici' UCM B-321 JXDG01000105 99.1% 95% 1459 0.0 P. zeae OE48.2T CP077090 99.0% 100% 1542 0.0 P. tensinigenes ZA5.3T CP077089 99.0% 100% 1542 0.0 P. helmanticensis OHA11T HG940537 99.0% 95% 1459 0.0 P. crudilactis UCMA 17988T WXVV01000001 99.0% 99% 1525 0.0 P. laurylsulfativorans AP3_22T MF554631 98.8% 95% 1459 0.0 4 P. umsongensis DSM 16611T NIWU01000003 98.8% 95% 1459 0.0 P. mohnii DSM 18327T FNRV01000001 98.6% 95% 1459 0.0 P. parafulva NBRC 16636T BBIU01000051 98.6% 95% 1459 0.0 P. mucoides P154aT LR797589 98.6% 80% 1232 0.0 P. fulva NBRC 16637T BBIQ01000036 98.6% 95% 1459 0.0 P. iridis P42T LR797554 98.5% 80% 1232 0.0 P. laurylsulfatiphila AP3_16T KY462012 98.5% 95% 1460 0.0 P. lutea DSM 17257T JRMB01000004 98.4% 95% 1459 0.0 P. defluvii WCHP16T KY979145 98.3% 95% 1459 0.0 P. asiatica RYU5T MH517510 98.3% 95% 1459 0.0 P. taiwanensis BCRC 17751T EU103629 98.3% 94% 1451 0.0 P. eucalypticola NP-1T MN238862 98.3% 92% 1423 0.0 P. soli F-279,208T HF930598 98.2% 92% 1421 0.0 P. turukhanskensis IB1.1T KP306892 98.2% 92% 1412 0.0 P. gessardii CFML 95-251T AF074384 98.2% 95% 1459 0.0 P. mucidolens DSM 19186T KX186963 98.2% 95% 1459 0.0 P. inefficax JV551A3T OPYN01000008 98.2% 95% 1459 0.0 P. agarici NCPPB 2289T AKBQ01000002 98.2% 95% 1459 0.0 P. rhizosphaerae IH5T AY152673 98.2% 95% 1459 0.0 P. plecoglossicida NBRC 103162T BBIV01000080 98.15% 95% 1459 0.0 P. monteilii NBRC 103158T BBIS01000088 98.2% 95% 1459 0.0 P. moorei RW10T AM293566 98.1% 95% 1459 0.0 P. entomophila L48T AY907566 98.1% 95% 1459 0.0 P. saponiphila DSM 9751T (FM208264) 96.5% 99% 1530 0.0 H. pertucinogena NBRC 14163T (AB680571) 94.5% 95% 1459 0.0 Table S2 Species Strain Accession ID Identity Cover Length E value P. gozinkensis IzPS32dT NZ_CP062253 98.2 99% 9873 0.0 P. glycinae MS586T NZ_CP014205 98.2 100% 9799 0.0 'P. kribbensis' 46-2 NZ_CP029608 97.9 99% 9799 0.0 P. allokribbensis IzPS23T NZ_CP062252 97.40 100% 9873 0.0 P. koreensis Ps9-14T NZ_JAAQYM010000010 97.0 99% 9795 0.0 P. atagonensis PS14T NZ_VXCA010000019 96.9 100% 9797 0.0 P. atacamensis M7D1T NZ_SSBS01000010 96.5 100% 9799 0.0 P. moraviensis CCM 7280T NZ_LT629788 96.4 100% 9799 0.0 P. granadensis LMG 27940T NZ_LT629778 96.3 100% 9799 0.0 P. baetica a390T NZ_PHHE01000001 96.1 99% 9786 0.0 P. crudilactis UCMA 17988T NZ_WXVV01000001 96.1 100% 9865 0.0 P. zeae OE48.2T NZ_CP077090 96.0 100% 9879 0.0 P. tensinigenes ZA 5.3T NZ_CP077089 96.0 100% 9879 0.0 P. neuropathica P155T NZ_JACOPX010000000 95.9 97% 9569 0.0 P. helmanticensis OHA11T SAMN04488483 95.9 100% 9796 0.0 P. hamedanensis SWRI65T NZ_CP012001 95.6 100% 9879 0.0 P. izuensis IzPS43_3003T NZ_WTFT01000001 94.9 100% 9799 0.0 P. vancouverensis ATCC 700688T NZ_RRZK01000005 94.9 99% 9799 0.0 P. jessenii DSM 17150T NZ_FNTC01000002 94.8 100% 9799 0.0 P. umsongensis DSM 16611T NZ_LT629767 94.7 100% 9799 0.0 P. laurylsulfatiphila AP3_16T NZ_NIRS01000001 94.7 99% 9800 0.0 P. laurylsulfativorans AP3_22T NZ_MUJK01000010 94.7 98% 9642 0.0 P. iridis P42T NZ_JACOPU010000001 94.5 97% 9572 0.0 P. reinekei Mt-1T NZ_LT629709 94.4 100% 9799 0.0 P. moorei RW10T NZ_FNKJ01000003 94.2 100% 9799 0.0 P. mohnii DSM 18327T NZ_FNRV01000002 94.2 100% 9799 0.0 P. mucoides P154aT NZ_JACOPW010000001 93.6 97% 9572 0.0 'P. batumici' UCM B-321 NZ_JXDG01000001 93.6 100% 9799 0.0 P. gessardii CFML 95-251T NZ_FNKR01000003 93.0 100% 9802 0.0 P. saponiphila DSM 9751T NZ_FNTJ00000000 92.6 99% 9870 0.0 P. mucidolens DSM 19186T NZ_LS483433 92.5 100% 9799 0.0 P. agarici NCPPB 2289T NZ_FOAR01000077 91.4 99% 9799 0.0 P. eucalypticola NP-1T NZ_CP056030 90.1 99% 9763 0.0 P. rhizosphaerae IH5T NZ_CP009533 89.3 99% 9796 0.0 P. plecoglossicida NBRC 103162T NZ_BBIV01000097 89.2 100% 9805 0.0 P. entomophila L48T CT573326 89.1 99% 9805 0.0 P. lutea DSM 17257T JRMB01000001 88.9 99% 9793 0.0 P. inefficax JV551A3T NZ_OPYN01000001 88.9 100% 9805 0.0 P. asiatica RYU5T NZ_BLJF01000001 88.9 100% 9805 0.0 P. monteilii NBRC 103158T NZ_BBIS01000132 88.8 100% 9805 0.0 P. soli F-279,208T NZ_CP009365 88.7 99% 9767 0.0 P. taiwanensis BCRC 17751T NZ_KE384450 88.7 100% 9797 0.0 P. parafulva NBRC 16636T NZ_BBIU01000055 - 88.3 100% 9802 0.0 NZ_BBIU01000000 P. fulva NBRC 16637T LAWW01000001 88.0 100% 9802 0.0 H. pertucinogena JCM 11590T NZ_BMNN00000000 84.1 86% 9814 0.0 Table S3 Type of Most similar Cluster Region Position MIBiG comparison metabolite known cluster similarity Fragin (Burkholderia cenocepacia H111) 1 Arylpolyene 675,175 - 718,779 APE Vf 40% Fellutamide B (Aspergillus nidulans FGSC A4) Showdomycin (Streptomyces showdoensis) Duramycin (Streptomyces cinnamoneus) 2 RiPP-like 1,652,937 - 1,661,494 - 0 Butyrivibriocin AR10 (Butyrivibrio fibrisolvens) Citrulassin E (Streptomyces glaucescens) strain FIT81T Sodorifen (Serratia plymuthica 4Rx13) NRPS 2-methylisoborneol (Pseudanabaena sp. dqh15) 3 2,318,719 - 2,394,454 Pyoverdin 20% Terpene 2-methylisoborneol (Streptomyces griseus subsp. griseus NBRC 13350) Lipopolysaccharide (Xanthomonas campestris pv. campestris) 4 RiPP-like 2,686,928 - 2,697,767 - 0 T3 toxin (Cylindrospermopsis raciborskii T3) Saxitoxin (Cylindrospermopsis raciborskii T3) 9 Gacamide A (Pseudomonas fluorescens Pf0-1) 5 NRPS 2,811,220 - 2,886,660 Lokisin 71% Bananamide (Pseudomonas fluorescens) Anikasin (Pseudomonas fluorescens) Massetolide A (Pseudomonas fluorescens SS101) 6 NRPS 2,893,410 -2,952,691 Rimosamide 28% Thalassospiramide A (Tistrella bauzanensis) Sevadicin (Paenibacillus larvae) Aerobactin (Xenorhabdus szentirmaii DSM 16338) Aerobactin (Pantoea ananatis) 7 Siderophore 3,443,252 - 3,462,180 - 0% Ochrobactin (Ochrobactrum anthropi) Desferrioxamine (Pantoea agglomerans) Jadomycin (Streptomyces venezuelae ATCC 10712) Cepacin A (Burkholderia ambifaria IOP40-10) 8 Betalactone 4,195,007 - 4,218,271 Fengycin 13% Bromopyrroles/bromophenols (Marinomonas mediterranea MMB-1) Erythreapeptin (Saccharopolyspora erythraea NRRL 2338) 9 Ranthipeptide 4,480,897 - 4,502,327 Pyoverdin 11% Bicereucin (Bacillus cereus SJ1) FR-900098 (Streptomyces rubellomurinus) Erythrochelin (Saccharopolyspora erythraea NRRL 2338) 10 NRPS 4,531,025 - 4,584,023 Pyoverdin 21% Anabaenopeptin NZ857/nostamide A (Nostoc punctiforme PCC 73102) monobactam (Agrobacterium tumefaciens) Tabtoxin (Pseudomonas syringae) Bacilysin (Bacillus sp. CS93) 11 NAGGN 4,768,261 - 4,783,034 - 0% 4-hydroxy-3-nitrosobenzamide (Streptomyces murayamaensis) Prodigiosin (Serratia sp.) Redox- 6,092,219 - 6,114,387 12 Lankacidin C 13% Prodigiosin (Serratia marcescens) cofactor Prodigiosin (Hahella chejuensis KCTC 2396) Fragin (Burkholderia cenocepacia H111) 1 NRPS-like 130.089 - 159.699 Fragin 37% Livipeptin (Streptomyces lividans 1326) P. gozinkensis IzPS32dT Aspergillic acid (Aspergillus flavus NRRL3357) Aryl polyene (Escherichia coli CFT073) 2 Arylpolyene 488,336 - 531,964 APE Vf 40% Aryl polyene (Xenorhabdus doucetiae) Caboxamycin (Streptomyces sp. NTK 937) 3 RiPP-like 1,472,919 - 1,481,478 - 0% Duramycin (Streptomyces cinnamoneus) Butyrivibriocin AR10 (Butyrivibrio fibrisolvens) Citrulassin E (Streptomyces glaucescens) Livipeptin (Streptomyces lividans 1326) NRPS-like 4 2,103,351 - 2,187,903 Pyoverdin 20% Fragin (Burkholderia cenocepacia H111) NRPS Fellutamide B (Aspergillus nidulans FGSC A4) Lipopolysaccharide (Xanthomonas campestris pv. campestris) 5 RiPP-like 2,415,999 - 2,426,838 - 0% T3 toxin (Cylindrospermopsis raciborskii T3) Saxitoxin (Cylindrospermopsis raciborskii T3) Gacamide A (Pseudomonas fluorescens Pf0-1) 6 NRPS 2,664,065 - 2,733,716 Lokisin 71% Bananamide (Pseudomonas fluorescens) Anikasin (Pseudomonas fluorescens) Massetolide A (Pseudomonas fluorescens SS101) 7 NRPS 2,741,718 - 2,802,189 Rimosamide 21% Thalassospiramide A (Tistrella bauzanensis) Sevadicin (Paenibacillus larvae) Ethylenediaminesuccinic acid hydroxyarginine (EDHA) 8 Siderophore 2,847,118 - 2,858,971 - 0% (Streptomyces avermitilis MA-4680 = NBRC 14893) Lipopolysaccharide (Xanthomonas campestris pv. campestris) streptonigrin (Streptomyces flocculus) Pseudopyronine (Pseudomonas putida) 9 Thiopeptide 3,394,589 - 3,424,725 - 0% Pyrazomycin (Streptomyces candidus) Thaxteramide (Jahnella sp. MSr9139) Jadomycin (Streptomyces venezuelae ATCC 10712) Cepacin A (Burkholderia ambifaria IOP40-10) 10 Betalactone 4,186,987 - 4,210,223 Fengycin 13% Bromopyrroles/bromophenols (Marinomonas mediterranea MMB-1) FR-900098 (Streptomyces rubellomurinus) 11 Ranthipeptide 4,485,491 - 4,506,921 Pyoverdin 11% Succinoglycan (Sinorhizobium meliloti) Exopolysaccharides (Burkholderia cenocepacia J2315) Erythrochelin (Saccharopolyspora erythraea NRRL 2338) 12 NRPS 4,539,277 - 4,592,278 Pyoverdin 20% Anabaenopeptin NZ857/nostamide A (Nostoc punctiforme PCC 73102) chloromyxamide (Myxococcus sp.) Tabtoxin (Pseudomonas syringae) 13 NAGGN 4,747,766 - 4,762,638 - 0% Bacilysin (Bacillus sp. CS93) 4-hydroxy-3-nitrosobenzamide (Streptomyces murayamaensis) Prodigiosin (Serratia sp.) Redox- 14 5,936,419 - 5,958,575 Lankacidin C 13% Prodigiosin (Serratia marcescens) cofactor Prodigiosin (Hahella chejuensis KCTC 2396) Tabtoxin (Pseudomonas syringae) Bacilysin (Bacillus sp. CS93) 1 NAGGN 202,379 - 217,152 - 0% 4-hydroxy-3-nitrosobenzamide (Streptomyces murayamaensis) Anabaenopeptin NZ857/nostamide A (Nostoc punctiforme P. glycinae MS586T PCC 73102) 2 NRPS 1,027,109 - 1,078,566 Pyoverdin 20% Monobactam (Agrobacterium tumefaciens) Aspirochlorine (Aspergillus oryzae RIB40) Massetolide A (Pseudomonas fluorescens SS101) 3 NRPS 2,076,149 - 2,134,645 Rimosamide 28% Sevadicin (Paenibacillus larvae) Curacomycin (Streptomyces curacoi) 4 NRPS 2,291,136 - 2,358,877 Lokisin 71% Bananamide (Pseudomonas fluorescens) Gacamide A (Pseudomonas fluorescens Pf0-1) Anikasin (Pseudomonas fluorescens) Lipopolysaccharide (Xanthomonas campestris pv. Bacillomycin campestris) alkylresorcinol (Streptomyces griseus subsp. 5 RiPP-like 2,532,324 - 2,541,936 20% D griseus NBRC 13350) Malleobactin (Burkholderia thailandensis E264) Prodigiosin (Serratia sp.) Redox- 6 2,973,621 - 2,995,777 Lankacidin C 13% Prodigiosin (Serratia marcescens) cofactor Prodigiosin (Hahella chejuensis KCTC 2396) Duramycin (Streptomyces cinnamoneus) butyrivibriocin 7 RiPP-like 4,555,244 - 4,566,131 - 0% AR10 (Butyrivibrio fibrisolvens) citrulassin E (Streptomyces Glaucescens) Showdomycin (Streptomyces showdoensis) 8 Betalactone 5,098,665 - 5,123,195 Fengycin 13% Jadomycin (Streptomyces venezuelae ATCC 10712) Cepacin A (Burkholderia ambifaria IOP40-10) Fragin (Burkholderia cenocepacia H111) 9 NRPS-like 5,205,531 - 5,235,101 Fragin 37% Livipeptin (Streptomyces lividans 1326) Aspergillic acid (Aspergillus flavus NRRL3357) Aryl polyene (Escherichia coli CFT073) 10 Arylpolyene 5,569,355 - 5,612,983 APE Vf 40% Aryl polyene (Xenorhabdus doucetiae) Caboxamycin (Streptomyces sp. NTK 937 2-methylisoborneol (Streptomyces griseus subsp. griseus NRPS- NBRC 13350) 11 6,231,816 -6,299,285 Pyoverdin 18% Terpene Bacilysin (Bacillus sp. CS93) Pentostatine/vidarabine (Streptomyces antibioticus) Table S4 1 2 3 NAD(P)(+) transhydrogenase (AB-specific) + + + Transport Branched-chain amino acid ABC transporter + + + Secretion and delivery Type VI secretion ATPase, ClpV1 + + + system Plant polymer Cupin + + + degradation/modification Peptidase M48 + + + AsnC family transcriptional regulator + + + AraC family transcriptional regulator + + + Transcriptional regulator Transcriptional regulator, DeoR family + + + Transcriptional regulator, LysR family + + + Glutathione S-transferase + + + S-(hydroxymethyl) glutathione dehydrogenase/class III + + + alcohol dehydrogenase Detoxification 2-dehydropantoate 2-reductase + + + Acetaldehyde dehydrogenase + - - Carbonate dehydratase + + + Aldehyde dehydrogenase + + + Redox potential L-lactate dehydrogenase + + + maintenance Malate dehydrogenase + + + 3-hydroxyisobutyrate dehydrogenase + + + ndvB + + + Plant cell-wall attachment ndvC + + + 1-aminocyclopropane-1-carboxylate deaminase + + + Hormones indole-3-glycerol phosphate synthase + + + Phosphate solubilization pqqA + + + pqqB + + + pqqC + + + pqqD + + + pqqE + + + pqqF + + + 2-isopropylmalate synthase + + + Other Diaminopimelate decarboxylase + + +