Frankia Torreyi Sp. Nov., an Actinobacterium Isolated From

1 Frankia torreyi sp. nov., an actinobacterium isolated from Comptonia peregrina that 2 effectively nodulates members of Myricaceae and Alnus species 3 4 5 Imen Nouioui1, Faten Ghodhbane-Gtari2, 3, Marlen Jando4, Louis S. Tisa5, Hans-Peter Klenk1, 6 Maher Gtari3*

7 8 1. School of Natural and Environmental Sciences, Newcastle University, Ridley Building 2, 9 Newcastle upon Tyne, NE1 7RU, United Kingdom. 10 2. Laboratoire Microorganismes et Biomolécules Actives, Université Tunis El Manar (FST), 11 2092 Tunis, Tunisia. 12 3. Institut National des Sciences Appliquées et de Technologie, Université Carthage, Centre 13 Urbain Nord, BP 676-1080 Tunis Cedex, Tunisia. 14 4. Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, 15 Inhoffenstraße 7B, 38124 Braunschweig, Germany. 16 5. Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire. 17 USA. 18 19 Corresponding author: Maher Gtari [email protected] 20 Section: Actinobacteria 21 Keywords: Frankia, symbiosis, chemotaxonomy, phenotyping 22

23 Running title: Description of Frankia torreyi sp. nov.

24 The journal’s contents category (New taxa-Actinobacteria)

25 The GenBank accession numbers of strain CpI1T for 16S rRNA gene and genome sequences 26 are MH423838 and JYFN00000000.1 respectively.

31 Abstract

32 Strain CpI1T is the first isolate of the genus Frankia that was obtained from Comptonia 33 peregrina root nodules in 1978. In this study, a polyphasic approach was performed to identify 34 the taxonomic position of strain CpI1T among the genus Frankia. It contained meso- 35 Diaminopimelic acid as the diagnostic diamino acid, and had galactose, glucose, mannose, 36 rhamnose, ribose and xylose as cell wall sugars. Polar lipids consisted of phosphatidylinositol

37 (PI), diphosphatidylglycerol (DPG), glycophospholipids (GPL1-3), phosphatidylglycerol (PG),

38 aminophospholipid (APL) and unknown phospholipids (PL1-2) and lipids (L). The predominant

39 menaquinone was MK-9 (H8), while the major fatty acid are iso-C16:0 and C17:1 ω 8c. The 16S 40 rRNA gene sequence identity varied from 97.4 to 99.6 % with the type strains of Frankia 41 species. Phylogenetic analyses based on 16S rRNA gene sequences and multilocus sequence 42 analysis (MLSA) using atp1, ftsZ, dnaK, gyrA and secA gene sequences showed that Frankia 43 alni ACN14aT is the closest phylogenetic relative. The genome size of strain CpI1T is 7.6 Mb 44 with a digital DNA G+C content of 72.4%. Digital DNA:DNA hybridization (dDDH) values 45 between strain CpI1T and its closest phylogenetic relative F. alni ACN14aT was 44.1% well 46 below the threshold of 70 % for distinguishing between bacterial genomic species. Based on 47 the phenotypic, phylogenetic and genomic data, strain CpI1T (= DSM44263T = CECT9035T) 48 warrants its classification as a type strain of a novel species for which the name Frankia torreyi 49 sp. nov. is proposed.

58 Introduction

59 The genus Frankia (Brunchorst 1886) of the family Frankiaceae (Becking 1970) and the order 60 Frankiales (Sen et al. 2014) covers soil-dwelling actinobacteria most of which fix nitrogen in 61 association with diverse dicotyledonous hosts, collectively named actinorhizal plants (Benson 62 and Silvester 1993; Schwencke and Carú 2001; Chaia et al. 2010). Based on single locus marker 63 of either the 16S rRNA (Normand et al. 1996), gyrB (Nouioui et al. 2011) or gln II (Normand 64 et al. 1996; Nouioui et al. 2011), the 16S-23S rRNA internal transcribed spacer (Ghodhbane- 65 Gtari et al. 2010), multilocus sequence analysis (MLSA) [atp1, ftsZ, dnaK, gyrA and secA] 66 (Gtari et al. 2015), or core genome (Tisa et al. 2016) phylogenies, Frankia strains are classified 67 into one of the four groups in line with the host plant specificity (Baker 1987). Cluster 1 68 encompasses strains infective towards Alnus (Betulaceae), Allocasuarina and Casuarina 69 (Casuarinaceae), and Myricaceae, while cluster 2 housed strains that are infective on 70 Coriariaceae, Datiscaceae, Dryadoideae, and Ceanothus (Rhamnaceae). Cluster 3 represents 71 Frankia associated with Elaeagnaceae, Colletieae (Rhamnaceae), Myricaceae, and 72 Gynmmostoma (Casuarinaceae). Additionally, cluster 4 includes atypical Frankia strains, 73 which are devoid from the abilities to fix nitrogen and/or to re-infect their host plants. Recently, 74 two to four species have been validly named within each of the four clusters. Frankia alni 75 (Nouioui et al. 2016), Frankia casuarinae (Nouioui et al. 2016) and Frankia canadensis 76 (Normand et al. 2018) of cluster 1. Frankia coriariae (Nouioui et al. 2017a), “Candidatus 77 Frankia datiscae” (Persson et al. 2011) and Candidatus Frankia californiensis (Normand et al. 78 2017) in cluster 2. Frankia elaeagni (Nouioui et al. 2016), Frankia discariae (Nouioui et al. 79 2017b) and Frankia irregularis (Nouioui et al. 2018a) within cluster 3. Cluster 4 contains 80 Frankia inefficax (Nouioui et al. 2017c), Frankia asymbiotica (Nouioui et al. 2017d) and 81 Frankia saprophytica (Nouioui et al. 2018b).

82 Strain CpI1T is the first isolate of the genus Frankia cultivated from the root nodules of 83 Comptonia peregrina (Callaham et al.1978). It is able to fulfil Koch’s postulates by inducing 84 effective root nodules on its original host plant and other members of Myricaceae and Alnus 85 species (Callaham et al. 1978; Lalonde 1979; Baker 1987; Torrey 1990). Strain CpI1T was 86 subjected to polyphasic taxonomic analysis, which underlined the phenotypic and phylogenetic 87 divergence of this strain from all other Frankia species which warrant its classification as the 88 type strain for a new species Frankia torreyi sp. nov.

89 Materials and methods

90 Strain CpI1T was maintained in basic propionate (BAP) medium (Murry et al. 1984)

91 supplemented with NH4Cl together with the type strains of all of the Frankia species (Table 1). 92 Four-week-old cultures were phenotypically characterized using GENIII microplates in an 93 Omnilog device (Biolog Inc., Haywood, USA). The ability of the strain to grow on a wide 94 range of carbon and nitrogen sources, under different pH and salinity conditions, and in the 95 presence of antibiotic or toxic compounds was tested as described previously (Nouioui et al. 96 2016). All of the tests were performed in duplicate. Cell wall sugars, diaminopimelic acids

97 (meso-A2pm; meso-diaminopimelate), fatty acids, menaquinone, and polar lipid patterns were 98 determined as described previously (Nouioui et al. 2016). 99 The 16S rRNA gene sequence obtained from a PCR-product was deposited in the GenBank

100 under accession number MH423838. Similarity matrices of the 16S rRNA gene sequences for 101 strain CpI1Tand the type strains of the Frankia species were performed using the genome to 102 genome distance calculator (GGDC) web server (Meier-Kolthoff et al. 2013a, b). Maximum- 103 likelihood (ML) and Maximum-parsimony (MP) trees were inferred using the same pipeline of 104 GGDC server as described previously (Nouioui et al. 2018a). Multilocus sequence analysis 105 (MLSA) based phylogeny was performed using 5 housekeeping genes (membrane-bound ATP 106 synthase, F1 sector, atpI; DNA gyrase subunit A, gyrA; tubulin-like GTP-binding protein, ftsZ; 107 ATPase secretory preprotein translocase, secA and chaperone Hsp70 in DNA biosynthesis, 108 dnaK). Gene sequences were retrieved from GenBank and concatenated to generate Maximum- 109 likelihood (ML) and Maximum-parsimony (MP) trees with the Mega 6.0 software (Tamura et 110 al. 2013) with 1000 bootstrap replicates. Digital DNA:DNA hybridization (dDDH) between 111 strains CpI1T and the rest of the Frankia species was calculated according to the pipeline of 112 GGDC (formula 2) (http://ggdc.dsmz.de/distcalc2.php). 113 114 Results and discussion 115 After 3-4 weeks incubation in BAP broth medium at 28ºC, strain CpI1T produced nonpigmented 116 cells with branched vegetative hyphae, vesicles and multilocular sporangia (Fig 1). Several 117 studies have confirmed the ability to fix nitrogen and to re-infect its host plant (Callaham et al. 118 1978; Lalonde 1979; Baker 1987; Torrey 1990). Based on several biochemical features, strain 119 CpI1T was easily distinguishable from its nearest relative F. alni ACN14aT. Comparatively, 120 strain CpI1T metabolised α-keto-butyric acid, d-gluconic acid, p-hydroxy-phenylacetic acid, 121 citric acid, bromo-succinic acid, α-keto-glutaricacid, D malic acid and acetic acid, while strain 122 ACN14aT oxidised methyl pyruvate and was found to metabolise D-glucose-6-phosphate, D- 123 fructose-6-phosphatea and guanidine hydrochloride better than strain CpI1T (Table 1).

T 124 Whole cell hydrolysates for strain CpI1 contained meso-A2pm, and had galactose, glucose, 125 mannose, xylose, ribose and rhamnose as cell wall sugars in concordance with previous reports 126 (Lechevalier and Ruan, 1984). The polar lipid pattern of strain CpI1T is as previously reported 127 (Lopez et al. 1983) and consisted of phosphatidylinositol (PI), diphosphatidylglycerol (DPG),

128 glycophospholipids (GPL1-3), phosphatidylglycerol (PG), aminophospholipid (APL) and

129 unknown phospholipids (PL1-2) and lipids (L) (Fig. S1). Apart from APL and PL, the polar lipid 130 composition of strain CpI1T is similar to F. alni type strain ACN14aT (Nouioui et al. 2016). The

131 predominant isoprenologue was MK-9(H8) (>20%), while its closest neighbour, strain T T 132 ACN14a , had MK-9(H8) and MK-9(H4). The major fatty acids (>15%) of strain CpI1 were

133 iso-C16:0 and C17:1 ω 8c in agreement with previous study (Tunfid et al. 1989) and which is a 134 similar profile as strain ACN14aT. 135 136 Pairwise 16S rRNA gene sequence similarities of strain CpI1T varied from 97.4-99.6 % with 137 the type strains of Frankia species (Table 2) and showed the highest value with F. alni ACN14aT 138 (99.6%). In Figure 2a, these two strains form together a subclade whithin cluster 2 (Alnus, 139 Allocasuarina, Casuarina and Myricaceae infective strain). The phylogenetic relatedness of 140 strain CpI1T to F. alni and members of cluster 1 was in line with the MLSA phylogeny (Fig 2b). 141 The dDDH values between strain CpI1T and F. alni ACN14aT (Table 2) was 44.1%, value well 142 below the threshold of 70% for delineation of a novel prokaryotic species (Wayne et al. 1987). 143 The genome size of CpI1T is 7.6 Mb with 72.4% of G+C content while its closest relative, F. 144 alni ACN14aT has 7.5Mb with 72.8 % of G+C content. 145 146 The phenotypic, phylogenetic and genomic data showed the divergence of strain CpI1T from 147 the type strains of other Frankia species. Therefore, it merits to be recognized as type strain of 148 a novel species for which the name Frankia torreyi sp. nov. is proposed. 149 150 Description of Frankia torreyi sp. nov. 151 Frankia torreyi (tor.re.yi N.L. fem. n. torreyi, named for John G. Torrey, who worked in 152 Harvard University, Petersham, MA USA, in recognition of his great contributions to Frankia 153 research including the isolation of the first Frankia strains) 154 Nitrogen-fixing, Gram-positive, aerobic, heterotrophic and chemoorganotrophic 155 actinobacterium characterised by three types of cell structures: substrate hyphae, multilocular 156 sporangia and vesicles. Optimal growth is observed after 3-4 weeks of incubation on BAP 157 medium at 28ºC with variable pH from 6.3 to 6.8. Able to metabolise propionate, α-keto-butyric

158 acid, D-gluconic acid, p-hydroxy-phenylacetic acid, citric acid, bromo-succinic acid, and α- 159 keto-glutaric acid and to grow in presence of aztreonam, lincomycin, minocycline, nalidixic 160 acid, troleandomycin and vancomycin.meso-Diaminopimelic acid, galactose, glucose, 161 mannose, rhamnose, ribose and xylose are detected in the whole cell hydrolysates, predominant 162 menaquinones (>80%) is MK-9 (H8), polar lipid profile consisted of phosphatidylinositol (PI),

163 diphosphatidylglycerol (DPG), glycophospholipids (GPL1-3), phosphatidylglycerol (PG),

164 aminophospholipid (APL) and unknown phospholipids (PL1-2) and lipids (L) as polar lipid. The

165 major fatty acids (>15%) are iso-C16:0 and C17:1 ω 8c. The size of the genome of the type strain 166 is 7.6 Mb and digital DNA G+C content is 72.4%. 167 The type strain CpI1T (=DSM 44263T = CECT 9035T) was isolated from the root nodules of 168 Comptonia peregrina (Callaham et al. 1978). 169 170 The GenBank/EMBL/DDBJ accession numbers of the genome and 16S rRNA gene sequence 171 of strain CpI1T are JYFN00000000.1 and MH423838 respectively.

172 Author contribution 173 MG conceived and designed the experiments, IN performed Biolog and chemotaxonomic 174 analysis, MJ helped in chemotaxonomic analysis, FGG and LST performed the genome 175 production, including genome assembly and GenBank submission, IN, FGG, LST, HPK and 176 MG analyzed the data. IN and MG wrote the paper: All authors read and approved the final 177 version of the manuscript. 178 Funding information no specific grant from any funding agency. 179 Conflicts of interest Authors have no conflict of interest to declare. 180 Acknowledgements This work was supported by Tunisian Ministry of Higher Education and 181 Scientific Research. We are grateful to Gabriele Pötter at DSMZ for helping in 182 chemotaxonomic analyses. 183 184 References

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193 Callaham C, Deltredici P, Torrey JG (1978) Isolation and Cultivation in vitro of the Actinomycete Causing 194 Root Nodulation in Comptonia. Science 199:899-902 195 Chaia EE, Wall L, Huss-Danell K (2010) Life in soil by actinorhizal root nodule endophyte Frankia. A 196 review Symbiosis 51:201-226 197 Ghodhbane-Gtari F, Nouioui I, Chair M, Boudabous A, Gtari M (2010) 16S-23S rRNA intergenic spacer 198 region variability in the genus Frankia. Microb Ecol60:487-495 199 Gtari M, Ghodhbane-Gtari F, Nouioui I, Ktari A, Hezbri K, Mimouni W, et al (2015) Cultivating the 200 uncultured:growing the recalcitrant cluster-2 Frankia strains. Sci Rep 5:13112 201 Lalonde M (1979) Immunological and ultrastructural demonstration of nodulation of the European 202 Alnus glutinosa (L.) Gaertn. host plant by an actinomycetal isolate from the North American 203 Comptonia peregrina (L.) Coult. root nodule. Bot Gaz 140:S35–S43 204 Lechevalier MP, Ruan JS (1984). Physiology and chemical diversity ofFrankia spp. isolated from nodules 205 ofComptonia peregrina (L.) Coult. andCeanothus americanus L. Plant Soil 78: 15-22. 206 Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M (2013a) Genome sequence-based species delimitation 207 with confidence intervals and improved distance functions. BMC Bioinform 14:60 208 Meier-Kolthoff JP, Göker M, Spröer C, Klenk H-P (2013b) When should a DDH experiment be mandatory 209 in microbial taxonomy? Arch Microbiol195:413-418 210 Murry MA, Fontaine MS, Torrey JG (1984) Growth kinetics and nitrogenase induction in Frankia sp. 211 HFP ArI3 grown in batch culture.Plant Soil 78:61-78 212 Normand N, Nouioui I, Pujic P, Fournier P, Dubost A, Klenk H-P et al (2018) Frankia canadensis sp. nov., 213 isolated from root nodules of Alnus incana subspecies rugosa growing in Canada. Int J Evol Syst 214 Microbiolo (accepted) 215 Normand P, Nguyen TV, Battenberg K, Berry AM, vanden Heuvel B, Fernandez MP et al (2017) Proposal 216 of ’Candidatus Frankia californiensis’, the uncultured symbiont in nitrogen-fixing root nodules of 217 a phylogenetically broad group of hosts endemic to western North America. Int J Syst Evol 218 Microbiol 67:3706–3715 219 Normand P, Orso S, Cournoyer B, Jeannin P, Chapelon C, Dawson J et al (1996) Molecular phylogeny of 220 the genus Frankia and related genera and emendation of the family Frankiaceae. Int J Syst 221 Bacteriol 46:1-9 222 Nouioui I, Ghodhbane-Gtari F, Beauchemin NJ, Tisa LS, Gtari M (2011) Phylogeny of members of the 223 Frankia genus based on gyrB, nifH and glnII sequences. Antonie Van Leeuwenhoek100:579-587 224 Nouioui I, Ghodhbane-Gtari F, Montero-Calasanz MD, Göker M, Meier-Kolthoff JP, Schumann P et al 225 (2016) Proposal of a type strain for Frankia alni (Woronin 1866) Von Tubeuf 1895, emended 226 description of Frankia alni, and recognition of Frankia casuarinae sp. nov. and Frankia elaeagni 227 sp. nov. Int J Syst Evol Microbiol 66:5201-5210 228 Nouioui I, Ghodhbane-Gtari F, Rohde M, Klenk H-P, Gtari M (2017a) Frankia coriariae sp. nov., an 229 infective and effective microsymbiont isolated from Coriaria japonica. Int J Syst Evol Microbiol 230 67:1266-1270 231 Nouioui I, Montero-Calasanz MDC, Ghodhbane-Gtari F, Rohde M, Tisa LS, Klenk H-P et al (2017b) 232 Frankia discariae sp. nov.: an infective and effective microsymbiont isolated from the root nodule 233 of Discaria trinervis. Arch Microbiol 199:641-647 234 Nouioui I, Ghodhbane-Gtari F, Montero-Calasanz MDC, Rohde M, Tisa LS, Gtari M et al (2017c) Frankia 235 inefficax sp. nov., an actinobacterial endophyte inducing ineffective, non nitrogen-fixing, root 236 nodules on its actinorhizal host plants. Antonie Van Leeuwenhoek 110:313-320 237 Nouioui I, Gueddou A, Ghodhbane-Gtari F, Rhode M, Gtari M, Klenk H-P (2017d) Frankia asymbiotica 238 sp. nov., a non infective actinobacterium isolated from Morella californica root nodule.Int J Syst 239 Evol Microbiol 67:4897-4901 240 Nouioui I, Ghodhbane-Gtari F, Rhode M, Sangal V, Klenk H-P, Gtari M (2018a) Frankia irregularis sp. 241 nov., an actinobacterium unable to nodulate its original host, Casuarina equisetifolia, but 242 effectively nodulate members of the actinorhizal Rhamnales. Int J Syst Evol Microbiol (accepted)

243 Nouioui I, Ghodhbane-Gtari F, Klenk H-P, Gtari M (2018b) Frankia saprophytica sp. nov. an atypical 244 non-infective (Nod–) and non-nitrogen fixing (Fix–) actinobacterium isolated from Coriaria 245 nepalensis root nodules. Int J Syst Evol Microbiol 68:1090-1095 246 Persson T, Benson DR, Normand P, vanden Heuvel B, Pujic P, Chertkov O et al (2011) Genome sequence 247 of "Candidatus Frankia datiscae" Dg1, the uncultured microsymbiont from nitrogen-fixing root 248 nodules of the dicot Datisca glomerata. J Bacteriol 193:7017–7018 249 Schwencke J, Carú M (2001) Advances in actinorhizal symbiosis: host plant-Frankia interactions, 250 biology, and applications in arid land reclamation. Arid Land Res Manag 15:285-327 251 Sen A, Daubin V, Abrouk D, Gifford I, Berry AM, Normand P (2014) Phylogeny of the class Actinobacteria 252 revisited in the light of complete genomes, the orders 'Frankiales' and Micrococcales should be 253 split into coherent entities. Proposal of Frankiales ord. nov., Geodermatophilales ord. nov., 254 Acidothermales ord. nov. and Nakamurellales ord. nov. Int J Syst Evol Microbiol 64:3821-3832 255 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics 256 Analysis version 6.0. Mol Biol Evol 30: 2725–2729 257 Tisa LS, Oshone R, Sarkar I, Ktari A, Sen A, Gtari M (2016) Genomic approaches toward understanding 258 the actinorhizal symbiosis: an update on the status of the Frankia genomes. Symbiosis 70:5-16 259 Torrey JG (1990) Cross-inoculation groups within Frankia and host-endosymbiont associations. In: 260 Schwintzer CR, Tjepkema JD (eds) The Biology of Frankia and Actinorhizal Plants. Academic Press, 261 Inc., New York, pp 83–106 262 Tunlid A, Schultz NA, Benson DR, Steele DB, White DC (1989). Differences in fatty acid composition 263 between vegetative cells and N2-fixing vesicles of Frankia sp. strain CpI1. PNAS 86:3399-3403. 264 Wayne LG, Brenner BD, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, et al (1987) Report of the 265 ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst 266 Bacteriol37:463-464 267 268 269 270 271 272 273 274 Figure Legends 275 276 Figure 1. Phase-contrast microscopy of strain CpI1T showing branched vegetative hyphae (h) 277 vesicle (v) (a) and extensive sporulation (s) (b). Bars indicated 10µm. 278 279 Figure 2. Maximum-likelihood phylogenetic tree based on almost complete 16S rRNA gene 280 sequences constructed using the GTR+GAMMA model. The numbers above the branches are 281 bootstrap support values greater than 60% for ML (left) and MP (right) (A). Multilocus 282 sequence analysis (MLSA) based Maximum-likelihood phylogeny was performed using 5 283 housekeeping genes (AtpI; GyrA; FtsZ; SecA and, DnaK). The numbers above the branches are 284 bootstrap support values greater than 60% for ML (left) and MP (right) (B). Bars indicate 285 estimated substitution per sequence position 286

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Table 1. Phenotypic and chemotaxonomic properties that distinguish strain CpI1T from the type strains of F. alni (ACN14aT), F. asymbiotica (M16386T), F. canadensis (ARgP5T), F. casuarinae (CcI3T), F. coriariae (BMG5.1T), F. elaeagni (BMG5.12T), F. discariae (BCU110501T),F. inefficax (EuI1cT),F. irregularis (G2T) and F. saprophytica (CN3T).

CpI1T ACN14AT M16386T ARgP5T CcI3T BMG5.1T BMG5.12T BCU110501T EuI1cT G2T CN3T

Colony colour white white white white white brown red yellow white red white greyish Vesicles/N2-fixation + + + + + + + + - + - Carbon source Dextrin - - - + - - - - + - - D-cellobiose - - + - - + + + - + + β-gentiobiose - - - - + - - - - - + D-glucose-6-phosphate w + - + - - + + - - + D-fructose-6-phosphate w + - + - + + + + - + α-hydroxy-butyricacid - - + - + + - + - w - β-hydroxy-butyricacid - - + - - - + - - - - α-keto-butyricacid + - + + + - + - - + + Aceto-aceticacid - - - + + + - - - w + Methyl pyruvate - + + - + + - - - + + D-gluconicacid + - + ------+ - L-lacticacid - - - + - + + + - + - L-malicacid + + ------w + D-malicacid w ------+ w + Citricacid + - - + - + - + + - + Bromo-succinicacid + ------+ + + + p-hydroxy-phenyl acetic acid + - - - + + - - - - - L-pyroglutamicacid + ------α-keto-glutaricacid + - - - - + - - + w +

Glucuronamide - - - - - + - - + w + Grow in presence of Acetic acid + - + + + + + + - + + 1% sodium lactate + + - + + + - + + + + Fusidic acid + + - + + - - + + w + Lithium chloride w + - + - - - + + w + Potassium tellurite + + + + + - - + + + + Sodium bromate + + + + - - - - + w + Nitrogen sources Guanidine hydrochloride w + - + - - - + + + - D-serine + + + + - - - - + w + Antibiotic resistance to# Lincomycin R R S R S S R R R R S Nalidixicacid R R R R R S S R R R R Minocycline and vancomycin R R S R R S S R R R R iso-C16:0, iso-C16:0, iso-C16:0, iso- iso- C18:1 iso-C16:0, C17:1ω8c, iso- iso- iso- C17:1 ω8c C17:1 ω8c C17:1 C16:0, C16:0, ω9c, C16:0, iso-C16:0, C16:0, C16:0, C16:0, Major fatty acids (>15%) C17:1 ω C17:1 C17:1ω8c C16 :0 C17:1ω8 C17:1 ω C17:1 ω8c C16:0 8c ω8c c, C17:0, 8c, C15:0 ω8c, C15:0 Predominant menaquinones MK9(H8) MK- MK- MK- MK- MK9(H6 MK- MK-9(H4) MK- MK- MK- (>20%) 9(H8), 9(H4), 9(H8) 9(H6), ), 9(H4), 9(H6), 9(H4); 9(H6) MK- MK- MK- MK9(H4 MK- MK- MK- 9(H4) 9(H6) 9(H8) ) 9(H6) 9(H4) 9(H6) Host plant origin Comptoni Alnus Morella Alnusi Casuar Coriaria Elaeagnu Discariatri Elaeag casuarin Coriaria a viridis californi ncana ina japonica sangustifo nervis nusumb a nepalen peregrina ssp.crispa ca ssp. cunnin lia elata equisetif sis rugosa ghami olia ana

Host plant range Betulacea Alnus, - Alnus Casuar Coriaria Elaeagna Colletieae, Elaeag Rhamna - e and Comptoni inacea ceae, ceae, Elaeagnace naceae, les Myricace a, Myrica e Datisca Colletieae ae Morella Morella ae (exclu ceae , Morella ding Gymos tom), Myrica ceae Genomic G+C content (%) 72.4 72.8 72.0 72.4 70.1 70.2 71.7 72.3 72.3 70.9 71.8

+, positive reaction; -, negative reaction; w, weak reaction; R, resistance; S, sensitive

Table 2. 16S rRNA gene sequence identities and dDDH values between strain CpI1T and the type strains of the nearest phylogenetic Frankia species. dDDH values are in % (lower left) and 16S rRNA gene sequence similarities are in % (upper right)

CpI1T ACN14aT ARgP5T CcI3T Frankia torreyi - 99.6 98.5 98.8 CPI1T Frankia alni 44.1 - 98.5 98.9 ACN14aT Frankia 25.6 25.7 - 98.9 canadensis ARgP5T Frankia 24.9 25.3 24.2 - casuarinae CcI3T