Non-Halophilic Endophytes Associated with the Euhalophyte Arthrocnemum Macrostachyum and Their Plant Growth Promoting Activity Potential M
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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 Received: 19 January 2018; Accepted: 22 August 2018 C FEMS 2018. All rights reserved. For permissions, please e-mail: [email protected] 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, incre- ment of mineral and nutrient availabilities, production of in- MATERIAL AND METHODS doleacetic acid (IAA) and ethylene, and phosphate solubiliza- tion (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);