International Journal of Systematic and Evolutionary Microbiology Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., plant growth promoting endophytes isolated from the halophyte plant Arthrocnemum macrostachyum. --Manuscript Draft-- Manuscript Number: IJSEM-D-18-00195R1 Full Title: Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., plant growth promoting endophytes isolated from the halophyte plant Arthrocnemum macrostachyum. Article Type: Taxonomic Description Section/Category: New taxa - Proteobacteria Keywords: Halomonadaceae, Odiel marshes, halophilic, PGPB, heavy metals Corresponding Author: Maria del Carmen Montero-Calasanz School of Biology, Newcastle University Newcastly upon Tyne, UNITED KINGDOM First Author: Salvadora Navarro-Torre Order of Authors: Salvadora Navarro-Torre Lorena Carro Ignacio D. Rodríguez-Llorente Eloísa Pajuelo Miguel Ángel Caviedes José Mariano Igual Susana Redondo-Gómez Maria Camacho Hans-Peter Klenk Maria del Carmen Montero-Calasanz Manuscript Region of Origin: SPAIN Abstract: Two endophytic bacteria (EAod3T and EAod7T) were isolated from the aerial part of plants of Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). Phylogenetic analysis based on 16S rRNA gene sequences indicated their affiliation to the genus Kushneria. 16S rRNA gene sequences of strains EAod3T and EAod7T showed the highest similarity with K. marisflavi DSM 15357T (99.0% and 97.6%, respectively). Digital DNA-DNA hybridization studies between the draft genomes of strain EAod3T and K. marisflavi DSM 15357T corresponded to 28.5% confirming the novel lineage of strain EAod3T in the genus Kushneria. Both strains were Gram staining-negative, aerobic and motile rods able to grow at 4-37ºC, at pH 5.0-8.0 and tolerate 0.5-25% NaCl (w/v). They presented ubiquinone Q9 and C16:0, C16:1ω7c/ C16:1ω6c and C18:1ω7c as the major fatty acids. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Based on the phenotypic and phylogenetic results, strains EAod3T (=CECT 9073T=LMG 29856T) and EAod7T (=CECT 9075T=LMG 29858T) are proposed as new representatives into the genus Kushneria, and the proposed names are K. phyllosphaerae sp. nov. and K. endophytica sp. nov., respectively. The whole genome sequence of strain EAod3T has a total length of 3.8 Mbp and a G+C content of 59.3 %. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document) Kushnerias sp. nov. - rev 2.doc 1 Kushneria phyllosphaerae sp. nov. and Kushneria endophytica 2 sp. nov., plant growth promoting endophytes isolated from 3 the halophyte plant Arthrocnemum macrostachyum. 4 5 Salvadora Navarro-Torre1, Lorena Carro2, Ignacio D. Rodríguez-Llorente1, 6 Eloísa Pajuelo1, Miguel Ángel Caviedes1, José Mariano Igual3, Susana 7 Redondo-Gómez4, Maria Camacho5, Hans-Peter Klenk2 & Maria del 8 Carmen Montero-Calasanz2* 9 1Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de 10 Sevilla, Calle Profesor García González, 2, 41012 Sevilla, Spain. 11 2School of Natural and Environmental Sciences (SNES), Newcastle University, 12 Newcastle upon Tyne, NE1 7RU, UK. 13 3Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de 14 Investigaciones Científicas (IRNASA-CSIC), c/Cordel de Merinas 40-52, 37008 15 Salamanca, Spain. 16 4Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de 17 Sevilla, 1095, 41012 Sevilla, Spain. 18 5IFAPA-Instituto de Investigación y Formación Agraria y Pesquera, Centro Las Torres- 19 Tomejil, Ctra. Sevilla-Cazalla de la Sierra, Km 12.2, 41200 Alcalá del Río, Sevilla, 20 Spain. 21 *Corresponding author: María del Carmen Montero-Calasanz, Tel.: +44 22 (0)191.208.4943 e-mail: [email protected] 23 Running title: Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov. 24 25 Subject category: New taxa (Proteobacteria) 26 27 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of 28 strains EAod3T and EAod7T are KU320856 and KU320860, respectively. The 29 GenBank/EMBL/DDBJ accession number for the draft genome sequence of strain 30 EAod3T is ONZI01. 31 32 Abstract 33 Two endophytic bacteria (EAod3T and EAod7T) were isolated from the aerial part of 34 plants of Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). 35 Phylogenetic analysis based on 16S rRNA gene sequences indicated their affiliation to 36 the genus Kushneria. 16S rRNA gene sequences of strains EAod3T and EAod7T showed 37 the highest similarity with K. marisflavi DSM 15357T (99.0% and 97.6%, respectively). 38 Digital DNA-DNA hybridization studies between the draft genomes of strain EAod3T 39 and K. marisflavi DSM 15357T corresponded to 28.5% confirming the novel lineage of 40 strain EAod3T in the genus Kushneria. Both strains were Gram staining-negative, 41 aerobic and motile rods able to grow at 4-37ºC, at pH 5.0-8.0 and tolerate 0.5-25% 42 NaCl (w/v). They presented ubiquinone Q9 and C16:0, C16:1ω7c/ C16:1ω6c and C18:1ω7c 43 as the major fatty acids. The predominant polar lipids were diphosphatidylglycerol, 44 phosphatidylglycerol and phosphatidylethanolamine. Based on the phenotypic and 45 phylogenetic results, strains EAod3T (=CECT 9073T=LMG 29856T) and EAod7T 46 (=CECT 9075T=LMG 29858T) are proposed as new representatives into the genus 47 Kushneria, and the proposed names are K. phyllosphaerae sp. nov. and K. endophytica 48 sp. nov., respectively. The whole genome sequence of strain EAod3T has a total length 49 of 3.8 Mbp and a G+C content of 59.3 %. 50 51 Keywords: Halomonadaceae, Odiel marshes, halophilic, PGPB, heavy metals 52 53 54 55 56 The genus Kusnheria is a monophyletic group belonging to the family Halomonadaceae 57 [1] in the class Gammaproteobacteria. It was first suggested by Sánchez-Porro et al. [2] 58 to accommodate the species Kusnheria avicenniae [2, 3], Kushneria indalinina [2, 4] 59 and Kushneria marisflavi [2, 5] previously classified into the genus Halomonas. At the 60 time of writing it comprises seven validly named species [6]: Kushneria aurantia (type 61 species) [2], K. avicenniae [2, 3], K. indalinina [2, 5], Kushneria konosiri [7], K. 62 marisflavi [2, 5, 8], Kushneria pakistanensis [9] and Kushneria sinocarnis [10]. 63 Representatives of the genus Kushneria are Gram-staining-negative, motile, non- 64 sporulating, halophilic and aerobic rods [2], presenting the ubiquinone Q9 as the major 65 respiratory quinone and C16:0, C18:1ω7c, C19:0cyclo ω8c and C12:0 3-OH as the 66 predominant fatty acids [2]. 67 Recently, endophytic strains EAod3T and EAod7T were isolated from the aerial part of 68 the halophyte plant Arthrocnemum macrostachyum in the Odiel marshes in Huelva 69 (Spain) (37º 13’N - 6º 57’O) (see Navarro-Torre et al. [11] for details). According to 70 Navarro-Torre et al. [11], both isolates were able to grow in presence of high 71 concentrations of heavy metals and salt, tolerate up to 20 mM Pb, 23 mM Ni and 3 M 72 NaCl, and hydrolyse starch (amylase activity) and DNA (DNAse activity). Additionally 73 the presence of the enzyme caseinase was also observed for strain EAod3T. They 74 besides showed in vitro plant growth promoting (PGP) properties such as siderophores 75 production, indolacetic acid (IAA) production, nitrogen fixation and phosphate 76 solubilisation, in absence and presence of moderate amounts of heavy metals [11]. 77 Strain EAod3T also demonstrated a promising biotechnological potential as plant 78 growth and bioremediation promoter when inoculated as part of a bacterial consortium 79 on plants of A. macrostachyum under greenhouse conditions [12]. 80 In the present study, a polyphasic characterization, following the minimal standards for 81 the family Halomonadaceae [13], was performed to determine the taxonomic status of 82 both strains. Based on phylogenetic and phenotypic data, strains EAod3T and EAod7T 83 are proposed as new species within the genus Kushneria, for which the names 84 Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov. are proposed. 85 86 Briefly, endophytes from the aerial part of A. macrostachyum were isolated on Tryptic 87 Soy Agar (TSA) supplemented with 0.3M NaCl and grown at 28ºC for 72 h and then, 88 different strains were sorted by colony morphology and colour (see Navarrro-Torre et 89 al. [11] for details]. Pure cultures were preserved at -80ºC in 15% glycerol. 90 DNA extraction, 16S rRNA gene sequences amplification and sequencing were carried 91 out as outlined by Navarro-Torre et al. [11]. 16S rRNA gene sequences were deposited 92 in GenBank/EMBL/DDBJ data library and aligned with corresponding sequences of 93 closely related type strains retrieved by Ez-Taxon-e service 94 (http://www.ezbiocloud.net/eztaxon) [14]. Genome from strain EAod3T was sequenced 95 and assembled by MicrobesNG company (Birmingham, United Kingdom) using 96 Illumina technology and a standard analysis pipeline. The closest available reference 97 genome was identified using Kraken [15], the quality of data was assessed mapping the 98 reads to this using BWA mem [16], de novo assembly of the reads was done using 99 SPAdes [17] maping the reads back to the resultant contigs, and again using BWA mem 100 to get more quality metrics. The whole draft genome was deposited in 101 GenBank/EMBL/DDBJ. RAST server v2.0 [18], QUAST v.4.6.3 software [19], 102