Non-Halophilic Endophytes Associated with the Euhalophyte Arthrocnemum Macrostachyum and Their Plant Growth Promoting Activity Potential M

FEMS Microbiology Letters, 365, 2018, fny208

doi: 10.1093/femsle/fny208 Advance Access Publication Date: 24 August 2018 Research Letter Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019

R E S E A RCH L E T T E R – Environmental Microbiology Non-halophilic endophytes associated with the euhalophyte Arthrocnemum macrostachyum and their plant growth promoting activity potential M. del R. Mora-Ruiz1,∗, C. Alejandre-Colomo1, T. Ledger2,B.Gonzalez´ 2, A. Orfila3 and R. Rossello-M´ ora´ 1

1Department of Ecology and Marine Resources, Mediterranean Institute for Advanced Studies (IMEDEA UIB-CSIC), Esporles, Spain, 2Facultad de Ingenier´ıa y Ciencias, Universidad Adolfo Iba´ nez˜ – Center of Applied Ecology and Sustainability, Santiago, Chile and 3Department of Marine Technologies, Operational Oceanography and Sustainability, Mediterranean Institute for Advanced Studies (IMEDEA UIB-CSIC), Esporles, Spain

∗Corresponding author: Department of Ecology and Marine Resources, Mediterranean Institute for Advanced Studies (IMEDEA UIB-CSIC), Esporles, Spain. Tel: +34 694469376; E-mail: [email protected] One sentence summary: Microbial diversity inhabiting the endosphere of halophytes. Editor: Paolina Garbeva

ABSTRACT

Numerous microbial taxa establish natural relations with plants, and especially endophytes can be relevant in the development and growth promotion of their host. In this work, we explore the diversity of non-halophilic microorganisms inhabiting the endosphere of the halophyte Arthrocnemum macrostachyum. A total of 1045 isolates were recovered using standard non-saline media, which clustered into 22 operational phylogenetic units (OPUs) including 7 putative new species and 13 OPUs not previously detected as endophytes. The more abundant isolates corresponded to close relatives of Kushneria indalinina/K. marisflavi, Providencia rettgeri, Pseudomonas zhaodongensis and Bacillus safensis, which made up to ∼ 62% of the total isolates. We also isolated OPUs not detected by the culture-independent approach reinforcing the need of culturing to reveal the microbial diversity associated with plants. Additionally, the plant growth promoting activity was evaluated by representative strains of the more abundant OPUs (total = 94 strains) including also some previously isolated halophiles from the same plants. Under both saline and non-saline conditions, some strains principally those affiliated to Paenibacillus borealis, Staphylococcus equorum, Salinicola halophilus and Marinococcus tarijensis, presented growth promoting activity in Arabidopsis thaliana, which was evaluated as an increment of weight and root length.

Keywords: Arthrocnemum macrostachyum; (non)-halophiles; operational phylogenetic unit; plant growth promoting activity

INTRODUCTION phytes (Newman and Reynolds 2005; Mapelli et al. 2013). En- dophytes are microorganisms that produce no apparent dam- The internal tissues of plants (endosphere) harbor microbial age to the plant and their role can be reflected positively as communities whose members are classified, depending on the contribution of nutrients, protection against pathogens and kind of interaction established, either as pathogens or endo- other plant growth promoting activities (PGPA; Rosenblueth

1 2 FEMS Microbiology Letters, 2018, Vol. 365, No. 19

and Mart´ınez-Romero 2006; Hardoim, van Overbeek and Elsas croorganisms (Mora-Ruiz et al. 2015, 2016). The results revealed 2008; Andreote, Azevedo and Araujo´ 2009; Mercado-Blanco and Chromohalobacter canadensis, Salinicola halophilus, Kushneria Lugtenberg 2014). As internal tissues of the plant can be a hos- indalinina and Rudaea cellulosilytica as the more relevant taxa tile environment (Mercado-Blanco and Lugtenberg 2014), spe- (Mora-Ruiz et al. 2015, 2016). In those studies, we recovered cific molecular communications are required between microor- by culture an important fraction of the richness observed Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019 ganisms and plants for the success of the colonization processes by massive sequencing (∼60%), but other abundant groups (Schikora, Schenk and Hartmann 2016). There are some studies (e.g. Halomonas meridiana, Pseudomonas seleniipraecipitans, Pseu- on colonization processes in different internal structures of the domonas alcaliphila, R. cellulosilytica and Cupriavius gilardii) plant (Lugtenberg, Chin-A-Woeng and Bloemberg 2002;Seghers were not cultured in part because we used culture media for et al. 2004; Rosenblueth and Mart´ınez-Romero 2006;Liet al. 2008; halophiles (from 5% to 30%). Therefore, to reveal whether Ryan et al. 2008), but, in general, the rhizosphere has attracted the remaining uncultured ∼40% could be non-halophile or most of the attention (Vessey 2003; Lugtenberg and Kamilova halotolerant organisms, we focused on the recovering of the 2009; Segura et al. 2009). After the colonization processes, the non-halophilic and halotolerant culturable bacteria inhabiting abundances of the endophytes are influenced by different fac- A. macrostachyum. To our knowledge, this is the first study tors being the more important the availability of nutrients for where the non-halophilic bacterial community structure from the microorganisms, the genotype and phase of development halophytes is analyzed with a large-scale culturing approach in- of the plant, the environmental conditions (Kuklinsky-Sobral cluding six different culture media and more than 1000 isolates. et al. 2005; Andreote, Azevedo and Araujo´ 2009) and the presence Additionally, to gain some idea on their putative roles associated of antibacterial substances such as terpenoids, benzoxazines, with plants we selected some halophilic strains from our previ- flavonoids and isoflavonoids (Hardoim, van Overbeek and Elsas ous studies where 814 strains had been isolated (Mora-Ruiz et al. 2008). 2015), and non-halophilic strains isolated here to analyze their As mentioned, most studies of endophytes and their rele- potential PGPA on the model plant Arabidopsis thaliana, a salt vance have been focused on bacteria of commercial plants and sensitive plant species. specifically, their capability to perform nitrogen fixation, increment of mineral and nutrient availabilities, production of in- MATERIAL AND METHODS doleacetic acid (IAA) and ethylene, and phosphate solubilization (Germida et al. 1988;Hureket al. 2002; Zinniel et al. 2002; Plant material sampling and treatment Kuklinsky-Sobral et al. 2004;Segherset al. 2004;Ryanet al. 2008; Doty et al. 2009;Manteret al. 2010; Weber, Videira and Simoes˜ Three plants identified as A. macrostachyum were collected in ◦ ◦ de Araujo 2013; Amaresan, Jayakumar and Thajuddin 2014). Salinas de’s Trenc. (39 21 03.3 N, 3 00 44.3 E) in Mallorca (Spain) However, these studies have been recently broadened including in January 2015. The samples were collected in individual sterile other plants such as metal-accumulating species (Belimov et al. zip-lock bags and brought to the laboratory to be immediately 2005) or plants that can survive in saline soils, halophytes (Sgroy processed. Forty grams of green stems from each plant were ex- et al. 2009; Glick and Glick 2012; Ruppel, Franken and Witzel cised, their surface sterilized and the plant tissues disaggregated 2013; Mora-Ruiz et al. 2015, 2016). Endophytes of halophytes have following the protocol described in Mora-Ruiz et al. (2015). The been considered an interesting alternative as biofertilizers for obtained plant extracts (EXs) were used as the endophyte inocu- plants under stress conditions (salt stress and drought) (Ruppel, lum (EI) for the culture-dependent approach (see next section). Franken and Witzel 2013; Mercado-Blanco and Lugtenberg 2014). Fragments of each treated plant were also used to prove the ef- In fact, to face the current world-wide high demand for food de- ficacy of the plant surface sterilization process. The sterile plant rived principally from the increment of the human population material was submerged in R2A media and gently shaken for 3 and the constant awareness of environmental damage and pro- min. This suspension was used as the sterile inoculum (SI) and tection, new agricultural practices are necessary towards a more consequently inoculated in the different culture media. sustainable and environmentally friendly agriculture (Glick and Glick 2012). Isolation of non-halophilic and halotolerant The study of endophytic communities has been principally heterotrophic bacteria addressed by culture-independent approaches, and in some cases including just a few isolates (Andreote, Azevedo and To achieve a high number of isolated microorganisms, six Araujo´ 2009;Manteret al. 2010; Weber, Videira and Simoes˜ de bacteriological culture media with different composition Araujo 2013). Although next generation sequencing techniques were selected: Reasoner’s 2A (R2A) medium for oligotrophs allow exploring the richness associated with plants, they also (Reasoner, Blannon and Geldreich 1979); Bacteria Screening limit the study of the colonizing microbial cells in detail (Zen- Media 523 (M523) for organisms present in plants (Kado and gler et al. 2002). Currently, large scale cultivation combined with Heskett 1970); a poor nitrogen medium (NP; Cavalcante and mass spectrometry techniques as the Whole-Cell Matrix As- Dobereiner 1988); a thioglycolate containing medium to de- sisted Laser Desorption Ionization–Time Of Flight Mass Spec- tect the presence of microaerophilic bacteria (TH; Leijh et al. trometry (WC MALDI-TOF MS) has opened the possibility to a 1984); and the modified Burk’s (BM) (Atlas 2010) and Rennie better understanding of the cultivable fraction of complex mi- (RMR; Rennie 1981) media to detect nitrogen-fixing microbes crobial ecosystems (Viver et al. 2015). WC MALDI-TOF MS identi- (Sturz and Kimpinski 2004). A seventh medium prepared fication has been recommended for large scale studies because with sterile EX was used trying to resemble plant conditions it is efficient, fast and cost-effective in comparison with other and nutrients. For preparation of EX medium, a total of 398 identification tools such as 16S rRNA sequencing (Munoz et al. g of sterilized and macerated plant tissue were mixed with 2011; Mora-Ruiz et al. 2015). 380 ml of sterile phosphate buffered saline 4x. This mix was In our previous studies, we explored the endophytic diver- disaggregated using ultrasound (Omni-Ruptor 400 Ultrasonic sity of Arthrocnemum macrostachyum using both 454 amplicon Homogenizer, Omni International Inc, Georgia, U.S.A.; 3 cycles pyrosequencing and culturing of moderately halophilic mi- of 60% and potency 10, 2 min) to lyse the vegetal cells. Then, the Mora-Ruiz et al. 3

samples were centrifuged (3300 xg, 5 min, room temperature). to the reconstructed tree using the parsimony tool in ARB. Eleven The supernatant was collected, and the pellet was discarded. almost complete sequences of good quality according to SILVA Centrifugation was repeated twice and the supernatant filtered standards, and belonging to the class Methanococci were used through 0.5 μm membranes (Sterivex Millipore, Darmstadt, as the archaeal outgroup in the phylogenetic reconstruction Germany). A new and last centrifugation was performed (51,275 of the different bacterial isolates. The conservational filter for Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019 xg, 15 min, room temperature) and the supernatant was fi- the bacterial domain implemented in the database was used in nally sterilized through filtration using 0.22 μm membranes all cases. The manual inspection of the tree topology allowed (Sterivex Millipore, Darmstadt, Germany). The final volume grouping the sequences in operational phylogenetic units (OPUs) of 160 mL of sterile EX was used to prepare the EX medium. with identity scores ranging from 88.6% to 100% with the closest To control the sterile conditions of this filtered EX, three relative type strains from the LTPs123 database (Yarza et al. 2010). of these plates were kept non-inoculated to verify absence For OPUs with no close relative type strain, the closest relative of microbial growth. sequence was selected from the SilvaRef NR99 (Quast et al. 2013). TheEIandSIfromeachofthethreeplantswereinoculated The sequences have been deposited at the EMBL repository un- in all media by triplicate using dilutions from 10−1 to 10−4.With der the accession numbers (LN651124-LN651155 and LS453314- the aim of recovering the maximum bacterial richness, two dif- LS453366). ferent selection methods were used. The first consisted on a vi- sual differentiation of colonies according to their morphology; DNA extraction and random amplification of at least four colonies per morphotype were thus selected. In polymorphic DNA (RAPD) fingerprints all cases, a minimum of 30 colonies was isolated by plant individual and medium. The second method was based on a ran- Five to six colonies within each OTU were randomly selected dom selection of colonies dividing the plates in quarters and and their DNA extracted as mentioned above. The RAPD finger- isolating all colonies from one single quarter. All the selected prints were generated using MasterMix 5PRIME (2.5X MasterMix, colonies were re-inoculated in the same original medium but 10 mM of primer, DNA ≤250 ng and distilled water), in a final complemented with 100 μL of Itraconazol (2 mg/mL; Bexal Far- volume of 25 μL. The primer used was RAPD1 in the same con- maceutica,´ Madrid, Spain) to minimize fungal contamination ditions as previously reported (Pena˜ et al. 2005). The amplicons (Mora-Ruiz et al. 2015). were visualized in 1.5% agarose gels stained with ethidium bromide.

WC MALDI-TOF MS screening, 16S rRNA gene Plant growth promoting activity tests sequencing and phylogenetic reconstruction Each strain was homogeneously inoculated at 1 × 104 colony 1 The identification of the isolates was performed using the tan- forming units (CFU) in 0.8% agar, MS /2 plates (Murashige and dem approach WC MALDI-TOF MS with 16S rRNA gene sequenc- Skoog 1962) and seeds (eight seeds per plate) of A. thaliana Col-0 ing and phylogenetic inference (Munoz et al. 2011;Viveret al. ecotype plants were sown in the agar surface. Plant growth in 2015). All MALDI-TOF MS spectra were manually supervised with vertically arranged plates was monitored during 21 d in thermo- the Maldi Biotyper v 3.2 (Bruker Daltonics, Germany) and com- regulated growth chambers at 22◦C with a 16h/8h light/dark pho- pared its database for their identification, obtaining scores from toperiod. Paraburkholderia phytofirmans strain PsJN was used as 1.703 to 2.379. The minimum sampling size was calculated for positive PGPA control since it is able to promote growth in several one plant by the sequential addition of MS in the dendrogram plants (Naveed et al. 2014;Trda´ et al. 2014), including A. thaliana until the Operational Taxonomic Unit (OTU) number tended to (Poupin et al. 2013). Root length was measured every 48 h using a the asymptotes. Each WC MALDI-TOF MS cluster was considered standard mm scale ruler, while plant fresh (FW) and dry weight as a single OTU, and for those that could not be identified with (DW) were determined after harvest at day 21. Individual Ara- this MS database, between 1 and 4 isolates were selected for 16S bidopsis plants were collected and weighed directly from agar rRNA gene sequencing. The DNA extraction was performed by plates for FW determinations, and then all eight plants from osmotic shock using MilliQ water followed by a thermal shock each treatment plate were pooled and kept for 24 h in a dry- ◦ ◦ consisting in three cycles of 100 Cto−20 C during 5 min. The ing oven at 50◦C. Total weight was then determined for each extract was used to amplify the 16S rRNA gene following the plant pool including one complete treatment plate (four repli- 5PRIME Mastermix protocol (2.5X master Mix, 10 mM of each cate plates per treatment were used). For those bacterial iso- primer, DNA ≤250 ng and distilled water), in a final volume of 50 lates that had a positive effect on the plant in the first assay, μL. The amplification was performed with the universal primers a second assay was carried out measuring the production of the for the Bacteria domain: GM3 (5 -AGAGTTTGATCATGGCTCAG-3 ) auxin IAA, that promotes cellular division and radicular ram- and S (5 -GGTTACCTTGTTACGACTT-3 ) following previously re- ification of the plant (Naveed et al. 2014), and the production ported conditions (Mora-Ruiz et al. 2015). The amplicons were vi- of the enzyme 1-aminocyclopropane-1-carboxylate deaminase sualized in 1% agarose gels and stained with ethidium bromide. (ACC deaminase), that promotes growth in plants influenced by PCR products were purified with MSBR Spin PCRapace (Invitek environmental stress, decreasing ethylene concentrations and GmbH, Berlin) and sent for sequencing to Secugen S.L. (Madrid, increasing those of ammonia in the rhizosphere, thus delaying Spain). the senescence of the plant (Glick 2014). Sequences were reviewed, corrected and assembled using Sequencher v 4.9 software (Genes Corporation, Michigan), and Statistical analysis and ecological indices aligned using the SINA software (SILVA; Quast et al. 2013)implemented in the ARB v 5.5 program package (Ludwig et al. The total bacterial abundances between plants and culture 2004). Alignments were finally manually improved. The phylo- media were compared using a permutation test (PERMANOVA; genetic reconstruction of the almost complete sequences was Anderson 2001) followed by a post-hoc pairwise permuta- performed using the RAxML algorithm also implemented in the tional group comparison because the data complied with the ARB software package, while partial sequences were later added precept of homoscedasticity, but not for normality even after 4 FEMS Microbiology Letters, 2018, Vol. 365, No. 19

Identification of the isolates from the endosphere of A. macrostachyum by a tandem MALDI-TOF MS and 16S rRNA gene sequence inference

A total of 1096 strains were isolated and analyzed using WC Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019 MALDI-TOF MS (Table S2, Supporting Information). The manual inspection of the spectra rendered a total of 1045 good quality spectra, which were subsequently used. The minimum number of strains isolated by plant/culture medium ranged from 23 in the EX medium to 35 in R2A medium (Table S3, Supporting Infor- mation). The spectra obtained were clustered in a dendrogram generating 36 OTUs, at a 700 distance (Fig. 2). A random selection of representative organisms from each OTU was used for sequencing and identification. The phylogenetic reconstruction based on the representative sequences of the 36 OTUs generated a total of 22 OPUs (Table 1). Only OPU8 was not recovered from the random sampling strategy, whereas four (OPU10, OPU14, OPU16 and OPU22) were not recovered from the morphology selection strategy (Table S2, Supporting Information). From the 22 OPUs, 10 (representing 70% of the total abundance of strains) affiliated with members

Figure 1. Boxplot of total abundances of cultivable microorganisms in the differ- of Proteobacteria, 4 (1.6% of abundance) with Actinobacteria ent media. Three groups can be distinguished (A, B and C) based on the permu- and 8 with Firmicutes (28.4% of abundance). When using the tation analysis. Error bars indicate standard deviation. CFU = Colony Forming 98.7% identity value as the lowest and conservative cutoff to Units. discriminate species (Stackebrandt and Ebers 2006), 13 OPUs affiliated with known species (Table 1 and Fig. 3) and seven with known genera but representing potential new species (OPU7, OPU11, OPU12, OPU13, OPU15, OPU20 and OPU 21). The most transformation. Additionally, the ecological indices of ‘real di- abundant OPUs in the collection were OPU1 (Kushneria indalin- versity’ of Jost (Jost 2006) were calculated in order to describe the ina/K. marisflavi; 232 isolates), OPU2 (Providencia rettgeri; with 144 communities in each culture media. With the aim to compare isolates), OPU5 (Pseudomonas zhaodongensis; with 126 isolates) the bacterial communities from each culture medium, a Morisita and OPU15 (Bacillus safensis; with 142 isolates). Additionally, index was calculated (Chao et al. 2008) and a multivariate cluster OPU1 and OPU15 were the unique OPUs isolated with all culture analysis (dendrogram) with Euclidian distance was also carried media (Table S2, Supporting Information). out. All the statistical analysis and ecological indices were The analysis of the abundances yields by culture media, obtained with the software R v 3.3 using the packages vegan comparing Morisita overlap indices, showed a higher similar- (Oksanen et al. 2015), vegetarian (Charney and Record 2015)and ity (94.5%) between NP and M523 media. Contrarily, RMR was spaa (Zhang 2013). For plant growth promotion assays and leaf markedly dissimilar from the rest (ranging between 2.0 to 13.2%; senescence, statistically significant differences between inoc- Table S4, Supporting Information). These results were corrob- ulated and non-inoculated control plants for each measured orated by a multivariate clustering where RMR was relatively parameter, were calculated using one way analysis of variance more similar to BM and EX, but distinct from the other samples (ANOVA). Tukey’s honestly significant difference tests, set with (Fig. S1 and Table S4, Supporting Information). The dissimilar- afixedP < 0.05 threshold value, were used to make comparisons ity of RMR medium was mainly due to a notable dominance of among different treatments. Homogeneity of variances and Pseudomonas spp.: OPU6 (P. graminis) and OPU7 (P. cichorii). In ad- normality tests were performed using the MINITAB Software, dition, the Jost index revealed R2A and NP as the culture media release 13.31 (Minitab Inc., State College, Pennsylvania). providing higher diversity, and RMR with lower diversity (Table S5, Supporting Information). RESULTS

Culturable cell counts of endophytic aerobic bacteria PGPA of isolates on A. thaliana of A. macrostachyum The isolates considered for the PGPA analysis were selected Sterile tests performed on the vegetal fragments did not show avoiding clonal varieties, which were tested using RAPD finger- growth in any culture media used after one month of incubation. prints generated for at least two organisms of each OTU. A total Therefore, the viable cell counts reported here were considered of 98 isolates were analyzed and we selected one representative true endophytes. Colonies were observed already after 72 h, be- from each banding pattern. The profiles showed between 2 and ing R2A, TH and M523 the media where growth was faster, with 9 bands, with product lengths ranging from 250 to 4000 bp (Sup- RMR medium showing the most delayed growth (>10 days). En- plementary Fig. S2). A total of 72 strains from 12 OPUs (1 to 6 per dophytic abundances ranged from 103 to 106 CFU g−1 (Fig. 1), and group; Table S6, Supporting Information) with different patterns the differences depended on the culture medium (P < 0.05; Ta- were finally selected for PGPA tests. In addition, 22 strains from ble S1, Supporting Information). Regarding CFU g−1 yields, three the five most representative moderate halophilic OPUs isolated groups were determined (Fig. 1), the media M523, R2A, TH and in a previous study (Mora-Ruiz et al. 2015) were also incorporated NP showed the highest yields; the media BM and EX showed in- to this part of the study (Supplementary Table S6). termediate yields; whereas the RMR culture medium exhibited The PGPA test performed with A. thaliana showed ten strains the lowest values. with higher levels than the control (200%), which were identified Mora-Ruiz et al. 5 Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019

Figure 2. Dendrogram of 1045 Main Spectra (MSP) showing the clustered OTUs with a cutoff of 700. Colours of the barcharts correspond to the plant sample number and the value inside indicates the number of isolates of each OTU, in each plant. The identification of the OTUs is based on the 16S rRNA gene affiliationven gi in Table S2 (Supporting Information).

as members of eight OPUs. From these strains, seven exhibited ination) were detected in any of these plant growth-promoting a significant increment of the root length in comparison with isolates (Fig. S3, Supporting Information). the non-inoculated control (P < 0.05; Fig. 4A). Strains showing a higher PGPA affiliated with P. borealis (strain Pb1), Staphylococ- DISCUSSION cus equorum (strain Se2), S. halophilus (strain Sh1)andMarinococ- cus tarijensis (strain Mt1). All four consistently increased root The endosphere of halophytes has been recently described as a length of A. thaliana both in non-saline and saline conditions. hypersaline environment being the habitat of several halophilic However, their performances under salt stress were all lower microorganisms (Sgroy et al. 2009; Mora-Ruiz et al. 2015). How- than the positive control P. phytofirmans PsJN (Fig. 4A). All the ten ever, here we explored the possibility of finding non-halophile strains analyzed increased fresh weight under non-saline con- strains in the endosphere of the halophyte A. macrostachyum by ditions (Fig. 4B), having P. borealis strain Pb1 a remarkable effect culture-dependent analysis as well as to explore the potential (350%). Again, their effects under salinity were much lower than PGPA of the strains isolated, as a way to understand the plant- those of P. phytofirmans PsJN, being non-significantly different microbe association. Although other studies addressed cultur- from the control, except for K. indalinina strain Ki1 (Fig. 4B). Dry ing of endophytic microbiota using different culture media, the weight measured for the inoculated plants showed a significant analyses and identification of the isolates is usually reduced to increase when inoculated with P. borealis strain Pb1, K. indalinina <200 strains (Li et al. 2012;Jacksonet al. 2013;Jinet al. 2014). strain Ki1 and S. halophilus strain Sh1 (Fig. 4C; P < 0.05). No pu- We evidenced that the use of different culture media resulted tative growth promotion activity functions, such as IAA produc- in a better recovery of the cultivable fraction of the plant as- tion and the ability to use ACC as a nitrogen source (ACC deam- sociated endophytic microbial communities. We could recover 6 FEMS Microbiology Letters, 2018, Vol. 365, No. 19 Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019 he corresponding similarity value with the closest strain and the 1.7 2 1.9 1 95.499.9 AJ312209 AY226508 6 1 99.998.9 AP00893499.3 AB009939 EU179327 56 19 3 99.596.4 EU660389 XC01920498.2 1 1 EU558284 7 88.6 X79450 8 97.6 Z76658 56 96.3 KF532968 1 99,6–100 EU216736 74 98.9–99.3 AY485792 29 98.9–99.7 AM941745/KF359966 235 97.3–98.6 AJ011321 34 99.6–99.999.7–99.891.4–99.0 KC456547 AJ575816 JQ762275 144 7 126 99.6–99.8 AF234854 143 98.0–99.9 Y11150 75 including the closest type species. Taxonomy of the closest type species Arthrocnemum macrostachyum FirmicutesFirmicutesFirmicutes BacilliFirmicutesFirmicutesFirmicutes Bacilli Bacilli Bacilli Bacillaceae Bacilli Bacilli Staphylococcaeae Carnobacteriaceae Paenibacillaceae Paenibacillaceae Marinilactibacillus piezotolerans Staphylococcus equorum Paenibacillaceae Bacillus safensis Paenibacillus borealis Paenibacillus Paenibacillus tundrae taichungensis FirmicutesFirmicutes Bacilli Bacilli Bacillaceae Staphylococcaeae Staphylococcus saprophyticcus subs Bacillus pumilus Proteobacteria Gammaproteobacteria Halomonadaceae Kushneria indalinina/K ProteobacteriaProteobacteria GammaproteobacteriaProteobacteria Alphaproteobacteriaia Alphaproteobacteriaia Moraxellaceae Sphingomonadaceae Rhodobacteraceae Sphingomonas desicabilis Acinetobacter johnsonii Paracoccus chinensis ProteobacteriaProteobacteria Gammaproteobacteria Gammaproteobacteria Enterobacteriaceae Enterobacteriaceae Providencia rettgeri Pantoe aeucrina ProteobacteriaProteobacteria Gammaproteobacteria Gammaproteobacteria Pseudomonadaceae Pseudomonadaceae Pseudomonas psychrotolerans Pseudomonas zhaodongensis ProteobacteriaProteobacteria Gammaproteobacteria Gammaproteobacteria Pseudomonadaceae Pseudomonadaceae Pseudomonas graminis Pseudomonas cichorii Actinobacteria Actinobacteria Micrococcaceae Nesterenkonia halotolerans Actinobacteria Actinobacteria Microbacteriaceae Curtobacterium flaccumfaciens pv Actinobacteria Actinobacteria Intrasporangiaceae Janibacter sanguinis Actinobacteria Actinobacteria Sanguibacteraceae Sanguibacter keddieii Endophytic OPUs detected in OPU8OPU9 33 32 OPU14OPU15 7, 8, 21 35, 36 OPU18OPU19OPU20OPU21 29 11,OPU22 21 9 10 13 OPU13OPU16 17 OPU17 27 22 OPUOPU1 OTU 1, 14, 29, 34 Phylum∗ ∗ Class Family Species Identity Accession number Number of isolates OPU10 6,OPU12 12, 16, 28 25 OPU2OPU3 15, 16, 4, 18, 26 31 OPU11 20 OPU4OPU5OPU6OPU7 6, 3, 12, 24 16, 28 5 2 Table 1. OPUs identified by WC MALDI-TOFaccession number MS is and also indicated. respective their In grey, values OPUs identified in theto genus Bruker level Daltonics database.and potential The new species. additional OPUs corresponded to 16S rRNA data and t Mora-Ruiz et al. 7 Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019

Figure 3. 16S rRNA sequence based phylogenetic reconstruction of representative isolates and their close relative type strains and additional reference sequences. Sequences of the class Methanococci were used as outgroup. Bolded OPUs includes potential new species. up to 22 OPUs that can be equalized to species (Mora-Ruiz et al. OPU18 (S. equorum). Both were the unique group cultivated here 2016), including the isolation of yet unreported taxa associated and in the study of Mora-Ruiz et al. (2015). As in our previ- with plants such as Paracoccus chinensis (OPU10) and Paenibacillus ous studies (Mora-Ruiz et al. 2015, 2016), some species of Pseu- taichungensis (OPU22) as well as some strains with PGPA poten- domonas were isolated from the endosphere of A. macrostachyum. tial. Moreover, we have isolated representatives of seven puta- However, here we isolated additional species of this genus (i.e. tive new species, including a potential new genus (OPU11, San- OPU4, OPU5, OPU6 and OPU7). In both studies, Pseudomonas guibacter keddieii) demonstrating that the large-scale cultivation members exhibited important abundances [∼22% and 25% of the is a valuable tool for retrieval of microbial novelty (Viver et al. total in (Mora-Ruiz et al. 2015, 2016) and this study, respectively] 2015). evidencing the relevance of these members in the endospheric From the 22 OPUs detected in this study, only eight (OPU1, halophyte environment. OPU2, OPU18, OPU4, OPU5, OPU6, OPU15 and OPU16) belonged Although our study was not successful in recovering the to genera that have been previously reported as endophytes in most abundant groups detected by culture-independent ap- halophytes, as well as other coastal plants (Sgroy et al. 2009; proaches (e.g. Rudaea cellulosilytica, Cupriavius gilardii, and P. se- Amaresan, Jayakumar and Thajuddin 2014; Mora-Ruiz et al. 2015, leniipraecipitans; Mora-Ruiz et al. 2015, 2016), here we cultured 2016). The OPU1 (K. indalinina/K. marisflavi) was here confirmed nearly 5.5% of the total richness observed by the amplicon as one of the more abundant taxa present in the endosphere sequencing strategy, additional to the already cultured 60% of A. macrostachyum (Mora-Ruiz et al. 2015, 2016)aswellasthe using media for moderate halophiles (Mora-Ruiz et al. 2015), 8 FEMS Microbiology Letters, 2018, Vol. 365, No. 19 Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019

Figure 4. (A) Root length, (B) fresh weight and (C) dry weight of Arabidopsis thaliana plants inoculated with potential growth promoting isolates, under saline or non- saline conditions. ∗ indicates significant differences with the non-inoculated control. Paenibacillus borealis (strain Pb1), Paenibacillus borealis (strain Pb2), Staphylococcus equorum (strain Se1), Staphylococcus equorum (strain Se1), Kushneria indalinina (strain Ki1), Pseudomonas zhaodongensis (strain Pz1), Bacillus safensis (strain Bs1), Salinicola halophilus (strain Sh1), Marinococcus tarijensis (strain Mt1), Pseudomonas. graminis (strain Pg1) and Paraburkholderia phytofirmans (strain PsJN). Data are normalized relative to control (non-inoculated) plants. + in axis X indicates potential new species. implying that we could isolate up to 66% of the total detected abundances, did not report an increment in bacterial richness richness. The non-halophilic part of the endophytic commu- (Eevers et al. 2015). nity can be inhabiting specific compartments in the internal Nearly 62% of the total isolates were included in four OPUs plant tissues where salinity is lower than the areas in where identified as K. indalinina/K. marisflavi (OPU1), P. rettgeri (OPU2), P. halophilic microorganisms are the dominant species (Mora-Ruiz zhaodongensis (OPU5) and B. safensis (OPU15). Members of these et al. 2016). In addition, we recovered yet unreported seven species had been reported with potential nitrogen fixation capa- groups as endophytes including OPU2 (P. rettgeri), OPU10 (P. bility (Mapelli et al. 2013), similarly to some species of the Pseu- chinensis) OPU12 (Janibacter sanguinis), OPU14 (N. halotolerans), domonas genus (Doty et al. 2009). The beneficial effects of these OPU17 (Staphylococcus saprophyticcus), OPU19 (Marinilactibacillus endophytic bacteria on their host plant can occur through simi- piezotolerans) and OPU22 (P. taichungensis). All have been pre- lar mechanisms described for plant growth-promoting rhizobac- viously found in sediments and soils (Newman and Reynolds teria as previously suggested by Kloepper, Ryu and Zhang (2004) 2005; Toffin et al. 2005;Ryanet al. 2008;Liet al. 2009;Singh, and Mercado-Blanco and Lugtenberg (2014). In accordance, our Singh and Dubey 2013;Aroraet al. 2014; Khessairi et al. 2014; results of PGPA activity revealed a positive effect of K. indalin- Mercado-Blanco and Lugtenberg 2014; Bringel and Couee´ 2015; ina/K. marisflavi isolates on A. thaliana despite no significant ACC Sharma, Kaul and Dhar 2015) and they could have entered deamination and IAA production was found. This is not nec- through the roots to colonize the endosphere of the plant essarily intriguing because, although these functions are usu- as hypothesized for other bacteria (Chi et al. 2005; Rosen- ally active and effective in plant growth promoting Proteobac- blueth and Mart´ınez-Romero 2006;Liuet al. 2014; Mora-Ruiz teria, such as P. phytofirmans (Ledger et al. 2016), the bacterium et al. 2016). used as positive control in this work, ACC deamination and IAA Here, we recovered up to 106 CFU g−1, which are lower yields production are not necessarily the unique functions explain- than when using seawater culture medium (SW) for isolation ing the observed plant growth effects. Other functions can be of moderate halophiles tested in previous studies, that reached linked to the PGPA of these strains such as the solubilization values up to 107 CFU g−1 in SW5% and SW15% (Mora-Ruiz of phosphates (Glick and Glick 2012; Mapelli et al. 2013), pro- et al. 2015). This order of magnitude difference confirms that duction of volatile compounds and some enzymes as pectinase endospheric environment might be dominated by moderately (Ledger et al. 2016). The latter highlights the need of using plant- halophilic bacteria (Mora-Ruiz et al. 2016). In this context, the bacteria assays to assess growth promotion potential, rather oligotrophic medium R2A produced the highest richness and di- than screening for ACC deamination and IAA production as a versity, a fact that is in accordance with nutrient-poor media first (sometimes unique) choice (Mayak, Tirosh and Glick 2004; being a better source for cultivable diversity (Aagot et al. 2001), Glick 2014). where dominant strains with slower growth rate are not out- Other strains mostly isolated from a previous work (Mora- competed by the fast-growing (Connon and Giovannoni 2002). Ruiz et al. 2015), such as: S. equorum strain Se2, S. halophilus strain The EX yielded three additional exclusively OPUs; i.e. P. chi - Sh1 and M. tarijensis strain Mt1, also exhibited important in- nensis (OPU10), N. halotolerans (OPU14) and B. pumilus (OPU16). crements of the root lengths on A. thaliana, with similar yields Similar studies using EXs, despite observing an increment of as the PGPA positive P. phytofirmans strain PsJN (Sessitsch et al. Mora-Ruiz et al. 9

2005). Although these strains, together with other isolated in Bringel F, Couee´ I. Pivotal roles of phyllosphere microorganisms the present work showed to be efficient under non-saline con- at the interface between plant functioning and atmospheric ditions, their effects decreased as salinity increases. This was trace gas dynamics. Front Microbiol 2015;6:486. not unexpected, as bacterial promotion of a particular growth Cavalcante VA, Dobereiner J. A new acid-tolerant nitrogen-fixing parameter under non-stressed conditions does not necessarily bacterium associated with sugarcane. Plant Soil 1988;108:23– Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019 imply a positive effect under saline stress. As we used the model 31. plant organism A. thaliana and the strains were isolated from a Chao A, Jost L, Chiang SC et al. A two-stage probabilistic ap- halophyte, the results of lower activity in saline conditions do proach to multiple-community similarity indices. Biometrics not imply a lack of effect on the plant host (A. macrostachyum). 2008;64:1178–86. It should be mentioned that the choice of performing the Charney N, Record S. Jost diversity measures for community PGPA analysis in the premiere model for plant biology (A. data. Package “vegetarian.” R Packag 23-3 2015: Available at: thaliana) is principally explained by the important features of cran.r-project.org/web/packages/veget. this plant including a short generation time, small size that Chi F, Shen S, Cheng H et al. Ascending migration of endophytic limited the requirement for growth facilities and prolific seed rhizobia, from roots to leaves, inside rice plants and assess- production through self-pollination. Additionally, the significant ment of benefits to rice growth physiology. Appl Environ Mi- amount of research reports related with this plant allows the crobiol 2005;71:7271–8. comparison of our results with other studies (Koornneef and Connon SA, Giovannoni SJ. High-throughput methods for Meinke 2010). culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl Environ Microbiol SUPPLEMENTARY DATA 2002;68:3878–85. Doty SL, Oakley B, Xin G et al. Diazotrophic endophytes of native Supplementary data are available at FEMSLE online. black cottonwood and willow. Symbiosis 2009;47:23–33. Eevers N, Gielen M, Sanchez-L´ opez´ A et al. Optimization of iso- ACKNOWLEDGEMENTS lation and cultivation of bacterial endophytes through addition of plant extract to nutrient media. Microb Biotechnol This study was funded by the Spanish Ministry of Economy 2015;8:707–15. projects CGL2012–39627-C03–03 and CLG2015 66686-C3–1-P, Germida JJ, Siciliano SD, De Freitas JR et al. Diversity of root- which were also both supported with European Regional De- associated bacteria associated with field-grown canola (Bras- velopment Fund (FEDER) funds. Merit Mora-RUIZ PhD was sup- sica napus L.) and wheat (Triticum aestivum L.). FEMS Microbiol ported by fellowship CVU 265934 from the National Council of Ecol 1988;26:43–50. Science and Technology (CONACyT), Mexico. The authors thank Glick BR. Bacteria with ACC deaminase can promote plant Salines de’s Trenc team for allowing the access to their instal- growth and help to feed the world. Microbiol Res 2014;169: lations and help in sampling and experiment organization. The 30–39. authors also acknowledge the economic support and uncondi- Glick BR, Glick BR. Plant growth-promoting bacteria: mecha- tional scientific interest of the Lipotrue SL and Deep Blue Sea nisms and applications. Scientifica 2012;2012:1–15. SL. Hardoim PR, van Overbeek LS, Elsas JD van. Properties of bacterial endophytes and their proposed role in plant growth. Conflict of interest. None declared. Trends Microbiol 2008;16:463–71. Hurek T, Handley LL, Reinhold-Hurek B et al. Azoarcus grass en- REFERENCES dophytes contribute fixed nitrogen to the plant in an uncul- turable state. MPMI 2002;15:233–42. Aagot N, Nybroe O, Nielsen P et al. An altered Pseudomonas Jackson CR, Randolph KC, Osborn SL et al. Culture dependent diversity is recovered from soil by using nutrient-poor and independent analysis of bacterial communities associ- Pseudomonas-selective soil extract media. Appl Environ Micro- ated with commercial salad leaf vegetables. BMC Microbiol biol 2001;67:5233–9. 2013;13:274. Amaresan N, Jayakumar V, Thajuddin N. Isolation and char- JinH,YangXY,YanZQet al. Characterization of rhizosphere and acterization of endophytic bacteria associated with chilli endophytic bacterial communities from leaves, stems and (Capsicum annuum) grown in coastal agricultural ecosystem. roots of medicinal Stellera chamaejasme L. Syst Appl Micro- 2014;13:247–55. biol 2014;37:376–85. Anderson MJ. A new method for non-parametric multivariate Jost L. Entropy and diversity. Oikos 2006;113:363–75. analysis of variance. Austral Ecol 2001;26:32–46. Kado CI, Heskett MG. Selective media for isolation of Agrobac- Andreote FD, Azevedo JL, Araujo´ WL. Assessing the diversity of terium, Corynebacterium, Erwinia, Pseudomonas,andXan- bacterial communities associated with plants. Braz J Microbiol thomonas. Phytopathology 1970;60:969–76. 2009;40:417–32. Khessairi A, Fhoula I, Jaouani A et al. Pentachlorophenol degra- Arora S, Patel PN, Vanza MJ et al. Isolation and characterization dation by Janibacter sp., a new actinobacterium isolated from of endophytic bacteria colonizing halophyte and other salt saline sediment of arid land. BioMed Res Int 2014;2014:296472, tolerant plant species from coastal Gujarat. Afr J Microbiol Res DOI: 10.1155/2014/296472. 2014;8:1779–88. Kloepper JW, Ryu C-M, Zhang S. Induced Systemic Resistance Atlas R. Handbook of Microbiological Media, Fourth Edition. Boca and Promotion of Plant Growth by Bacillus spp. Phytopathology Raton: CRC Press, 2010. 2004;94:1259–66. Belimov AA, Hontzeas N, Safronova VI et al. Cadmium-tolerant Koornneef M, Meinke D. The development of Arabidopsis as a plant growth-promoting bacteria associated with the roots model plant. Plant J 2010;61:909–21. of Indian mustard (Brassica juncea L. Czern.). Soil Biol Biochem Kuklinsky-Sobral J, Araujo´ WL, Mendes R et al. Isolation and 2005;37:241–50. characterization of soybean-associated bacteria and their 10 FEMS Microbiology Letters, 2018, Vol. 365, No. 19

potential for plant growth promotion. Environ Microbiol growth and colonization of maize by Burkholderia phytofir- 2004;6:1244–51. mans PsJN. Ann Microbiol 2015;65:1381–9. Kuklinsky-Sobral J, Araujo´ WL, Mendes R et al. Isolation and char- Newman LA, Reynolds CM. Bacteria and phytoremediation: acterization of endophytic bacteria from soybean (Glycine new uses for endophytic bacteria in plants. Trends Biotechnol max) grown in soil treated with glyphosate herbicide. Plant 2005;23:6–8. Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019 Soil 2005;273:91–9. Oksanen J, Blanchet FG, Kindt R et al. Vegan: Commu- Ledger T, Rojas S, Timmermann T et al. Volatile-mediated ef- nity Ecology Package. R Package Version 2.2-1. 2015, fects predominate in Paraburkholderia phytofirmans growth http://CRAN.R-project.org/package=vegan (20 June 2017, promotion and salt stress tolerance of Arabidopsis thaliana. date last accessed). Front Microbiol 2016;7:1838. Pena˜ A, Valens M, Santos F et al. Intraspecific comparative analy- Leijh PC, van Zwet TL, ter Kuile MN et al. Effect of thioglyco- sis of the species Salinibacter ruber. Extremophiles 2005;9:151– late on phagocytic and microbicidal activities of peritoneal 61. macrophages. Infect Immun 1984;46:448–52. Poupin MJ, Timmermann T, Vega A et al. Effects of the Li H-F, Qu J-H, Yang J-S et al. Paracoccus chinensis sp. nov., iso- plant growth-promoting bacterium Burkholderia phytofir- lated from sediment of a reservoir. Int J Syst Evol Microbiol mans PsJN throughout the Life Cycle of Arabidopsis thaliana. 2009;59:2670–4. PLoS One 2013;8:e69435. Li JH, Wang ET, Chen WF et al. Genetic diversity and potential Quast C, Pruesse E, Yilmaz P et al. The SILVA ribosomal RNA gene for promotion of plant growth detected in nodule endophytic database project: improved data processing and web-based bacteria of soybean grown in Heilongjiang province of China. tools. Nucleic Acids Res 2013;41:D590–6. Soil Biol Biochem 2008;40:238–46. Reasoner DJ, Blannon JC, Geldreich EE. Rapid seven-hour fecal Li L, Sinkko H, Montonen L et al. Biogeography of symbiotic coliform test. Appl Environ Microbiol 1979;38:229–36. and other endophytic bacteria isolated from medicinal Gly- Rennie RJ. A single medium for the isolation of acetylene- cyrrhiza species in China. FEMS Microbiol Ecol 2012;79:46–68. reducing (dinitrogen-fixing) bacteria from soils. Can J Micro- Liu J, Liu S, Sun K et al. Colonization on root surface by a biol 1981;27:8–14. phenanthrene-degrading endophytic bacterium and its ap- Rosenblueth M, Mart´ınez-Romero E. Bacterial endophytes and plication for reducing plant phenanthrene contamination. their interactions with hosts. Mol Plant Microbe Interact PLoS One 2014;9:e108249. 2006;19:827–37. Ludwig W, Strunk O, Westram R et al. ARB: A software environ- Ruppel S, Franken P, Witzel K. Properties of the halophyte micro- ment for sequence data. Nucleic Acids Res 2004;32:1363–71. biome and their implications for plant salt tolerance. Func- Lugtenberg B, Kamilova F. Plant-growth-promoting rhizobacte- tional Plant Biol 2013;40:940–51. ria. Annu Rev Microbiol 2009;63:541–56. Ryan RP, Germaine K, Franks A et al. Bacterial endophytes: Lugtenberg BJJ, Chin-A-Woeng TFC, Bloemberg GV. Microbe- recent developments and applications. FEMS Microbiol Lett plant interactions: Principles and mechanisms. Antonie van 2008;278:1–9. Leeuwenhoek, Int J Gen Mol Microbiol 2002;81:373–83. Schikora A, Schenk ST, Hartmann A. Beneficial effects of Manter DK, Delgado JA, Holm DG et al. Pyrosequencing reveals bacteria-plant communication based on quorum sensing a highly diverse and cultivar-specific bacterial endophyte molecules of the N -acyl homoserine lactone group. Plant Mol community in potato roots. Microb Ecol 2010;60:157–66. Biol 2016;90:605–12. Mapelli F, Marasco R, Rolli E et al. Potential for plant growth Seghers D, Wittebolle L, Top EM et al. Impact of agricultural prac- promotion of rhizobacteria associated with Salicornia grow- tices on the Zea mays L. endophytic community. Appl Environ ing in Tunisian hypersaline soils. BioMed Research International Microbiol 2004;70:1475–82. 2013;2013:248078, DOI: 10.1155/2013/248078. Segura A, Rodr´ıguez-Conde S, Ramos C et al. Bacterial responses Mayak S, Tirosh T, Glick BR. Plant growth-promoting bacteria and interactions with plants during rhizoremediation. Microb confer resistance in tomato plants to salt stress. Plant Physiol Biotechnol 2009;2:452–64. Biochem 2004;42:565–72. Sessitsch A, Coenye T, Sturz AV et al. Burkholderia phytofir- Mercado-Blanco J, Lugtenberg B. Biotechnological applications mans sp. nov., a novel plant-associated bacterium of bacterial endophytes. Curr Biotechnol 2014;3:60–75. with plant-beneficial properties. Int J Syst Evol Microbiol Mora-Ruiz MDR, Font-Verdera F, D´ıaz-Gil C et al. Moderate 2005;55:1187–92. halophilic bacteria colonizing the phylloplane of halophytes Sgroy V, Cassan´ F, Masciarelli O et al. Isolation and charac- of the subfamily Salicornioideae (Amaranthaceae). Syst Appl Mi- terization of endophytic plant growth-promoting (PGPB) or crobiol 2015;38:406–16. stress homeostasis-regulating (PSHB) bacteria associated to Mora-Ruiz MDR, Font-Verdera F, Orfila A et al. Endophytic micro- the halophyte Prosopis strombulifera. Appl Microbiol Biotechnol bial diversity of the halophyte Arthrocnemum macrostachyum 2009;85:371–81. across plant compartments. FEMS Microbiol Ecol Sharma T, Kaul S, Dhar MK. Diversity of culturable bacte- 2016;92:1–10. rial endophytes of saffron in Kashmir, India. SpringerPlus Munoz R, Lopez-L´ opez´ A, Urdiain M et al. Evaluation of matrix- 2015;4:661. assisted laser desorption ionization-time of flight whole cell Singh A, Singh M, Dubey S. Changes in Actinomycetes commu- profiles for assessing the cultivable diversity of aerobic and nity structure under the influence of Bt transgenic brinjal moderately halophilic prokaryotes thriving in solar saltern crop in a tropical agroecosystem. BMC Microbiol 2013;13:122. sediments. Syst Appl Microbiol 2011;34:69–75. Stackebrandt E, Ebers J. Taxonomic parameters revisited: taen- Murashige T, Skoog F. A revised medium for rapid growth ished gold standards. Microbiol Today 2006;33:152–5. and bio assays with tobacco tissue cultures. Physiol Plant Sturz AV, Kimpinski J. Endoroot bacteria derived from marigolds 1962;15:473–97. (Tagetes spp.) can decrease soil population densities of Naveed M, Qureshi MA, Zahir ZA et al. L-Tryptophan-dependent root-lesion nematodes in the potato root zone. Plant Soil biosynthesis of indole-3-acetic acid (IAA) improves plant 2004;262:241–9. Mora-Ruiz et al. 11

Toffin L, Zink K, Kato C et al. Marinilactibacillus piezotolerans sp. Weber OB, Videira SS, Simoes˜ de Araujo L. Identifica- nov., a novel marine lactic acid bacterium isolated from deep tion of culturable endophytes in Champaka pineap- sub-seafloor sediment of the Nankai Trough. Int J Syst Evol ple grown in an organic system. Afr J Agric Res Microbiol 2005;55:345–51. 2013;8:3422–30. Trda´ L, Fernandez O, Boutrot F et al. The grapevine flagellin Yarza P, Ludwig W, Euzeby´ J et al. Update of the all-species living Downloaded from https://academic.oup.com/femsle/article-abstract/365/19/fny208/5078864 by Conicyt / Secretaria Ejecutiva de Cincel user on 07 January 2019 receptor VvFLS2 differentially recognizes flagellin-derived tree project based on 16S and 23S rRNA sequence analyses. epitopes from the endophytic growth-promoting bacterium Syst Appl Microbiol 2010;33:291–9. Burkholderia phytofirmans and plant pathogenic bacteria. New Zengler K, Toledo G, Rappe M et al. Nonlinear partial differential Phytol 2014;201:1371–84. equations and applications: Cultivating the uncultured. Proc Vessey JK. Plant growth promoting rhizobacteria as biofertilizers. Natl Acad Sci 2002;99:15681–6. Plant Soil 2003;255:571–86. Zhang J. spaa: SPecies association analysis. R package version Viver T, Cifuentes A, D´ıaz S et al. Diversity of extremely 0.2.1. 2013. halophilic cultivable prokaryotes in Mediterranean, Atlantic Zinniel DK, Lambrecht P, Harris NB et al. Isolation and charac- and Pacific solar salterns: Evidence that unexplored sites terization of endophytic colonizing bacteria from agronomic constitute sources of cultivable novelty. Syst Appl Microbiol crops and prairie plants. Appl Environ Microbiol 2002;68:2198– 2015;38:266–75. 208.