Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 248078, 13 pages http://dx.doi.org/10.1155/2013/248078 Research Article Potential for Plant Growth Promotion of Rhizobacteria Associated with Salicornia Growing in Tunisian Hypersaline Soils Francesca Mapelli,1 Ramona Marasco,1 Eleonora Rolli,1 Marta Barbato,1 Hanene Cherif,2 Amel Guesmi,2 Imen Ouzari,2 Daniele Daffonchio,1 and Sara Borin1 1 DeFENS,DepartmentofFood,EnvironmentandNutritionalSciences(DeFENS),UniversityofMilan,ViaCeloria2, 20133 Milan, Italy 2 Laboratory of Microorganisms and Active Biomolecules, University of Tunis El Manar, Campus Universitaire, 2092 Tunis, Tunisia Correspondence should be addressed to Sara Borin; [email protected] Received 15 March 2013; Revised 30 April 2013; Accepted 3 May 2013 Academic Editor: George Tsiamis Copyright © 2013 Francesca Mapelli et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Soil salinity and drought are among the environmental stresses that most severely affect plant growth and production around the world. In this study the rhizospheres of Salicornia plants and bulk soils were collected from Sebkhet and Chott hypersaline ecosystems in Tunisia. Depiction of bacterial microbiome composition by Denaturing Gradient Gel Electrophoresis unveiled the occurrence of a high bacterial diversity associated with Salicornia root system. A large collection of 475 halophilic and halotolerant bacteria was established from Salicornia rhizosphere and the surrounding bulk soil, and the bacteria were characterized for the resistance to temperature, osmotic and saline stresses, and plant growth promotion (PGP) features. Twenty Halomonas strains showed resistance to a wide set of abiotic stresses and were able to perform different PGP activities in vitro at 5% NaCl, including ammonia and indole-3-acetic acid production, phosphate solubilisation, and potential nitrogen fixation. By using a gfp-labelled strain it was possible to demonstrate that Halomonas is capable of successfully colonising Salicornia roots in the laboratory conditions. Our results indicated that the culturable halophilic/halotolerant bacteria inhabiting salty and arid ecosystems have a potential to contribute to promoting plant growth under the harsh salinity and drought conditions. These halophilic/halotolerant strains could be exploited in biofertilizer formulates to sustain crop production in degraded and arid lands. 1. Introduction Among abiotic stresses soil salinity is one of the strongest factors affecting plant growth and yield7 [ ]. Conditions of The influence of microbes on plant fitness has been rec- high salt concentrations in the soil are very frequent in arid ognized both in conventional and extreme habitats, where and semiarid regions on Earth, where different halophytic the ability of rhizobacteria to facilitate plant adaptation and species can be found. Halophytes have been proposed as promote growth and productivity has been reported [1–6]. key players for saline soils reclamation [8], phytoremediation Root-associated bacteria can promote plant growth by direct of hydrocarbon and heavy metals polluted saline soils [9, and indirect mechanisms, the former including nutrient 10], and forage and oil seed production [11, 12]. Salicornia fixation and solubilisation and phytohormones synthesis. (Chenopodiaceae) is a subcosmopolitan plant genus com- Indirect activities include biocontrol, the ability to reduce or prising annual species strictly occurring in salty environ- avoid the harmful effects of phytopathogens. Both the host ments and widespread in several countries, including those plant and its associated microbiome gain an evolutionary of the Mediterranean basin. Salicornia densely colonises advantage to survive under harsh conditions by establishing different areas of southern Tunisia, including Sebkhet and tight interplays. Chott ecosystems, dominated by extreme values of aridity 2 BioMed Research International and soil salinity. Intense evaporation rates render Sebkhet and plot where salt crusts were absent (BDV4-S4, BDV4-S5, and Chott asdrysaltlakeswhichareinhospitableformostofthe BDV4-S6). Replicates of bulk soils were also sampled from organisms. the site BDV4 (presence of salt crust: BDV4-B1; absence The manipulation of natural resources to increase plant of salt crust: BDV4-B4). Rhizospheric soil was defined as productivity in lands traditionally considered unsuitable for soil particles tightly adhering to roots (1–3 mm) after gently agriculture is a challenging but necessary task, in the light shaking. Bulk soil was collected as control about 2 m far of the increasing world population and the need for food from any vegetation. Rhizosphere and bulk soils will be, production [13]. The efforts which aimed to the production respectively, indicated in the text with the codes S and B. of salt-resistant crops include conventional breeding, marker- Soil samples were collected using sterile spoons and stored ∘ ∘ assisted selection, and the creation of transgenic plants and in sterile bags at −20 C for molecular analyses and at 4 Cfor are nowadays focusing also on the halophyte potential to microbiological isolation. Soil salinity was measured with a guarantee a suitable food production in a salinized planet hand refractometer (Atago, Tokyo, Japan) after the extraction [14]. Different works in the last years highlighted the impor- of pore water from approximately 2 g of soil. tance of plant growth promoting bacteria in facilitating salt tolerance in plants devoted to food production [3, 7, 15, 16], and few reports emphasized the role of PGP bacteria 2.2. Metagenome Extraction and 16S rRNA Amplification. associated with Salicornia spp. [17–22]. The investigation of DNA was extracted from 0.5 g of soil using the protocol the rhizobacterial community associated to plants naturally established by Schbereiter-Gurtner et al. [25]. adapted to cope with extreme saline conditions might lead to DNA was quantified using NanoDrop 1000 spectropho- several knowledge outputs: (i) the understanding of the plant- tometer (Thermo Scientific, Waltham, MA, USA). microbe interaction under saline conditions, (ii) definition Bacterial 16S rRNA gene fragments (∼550 bp) were PCR of the mechanisms underlying plant growth with promotion amplified using primers 907R (3 -CCGTCAATTCCTTTG- under the salinity stress, and (iii) identification of bacterial AGTTT-5 ) and GC-357F (3 -CCTACGGGAGGCAGCAG- strains to design biological fertilizers exploitable for agricul- 5 with a 5 -end GC-clamp) targeting a portion of the 16S ture in arid and saline lands. To achieve the best results in rRNA gene that include the hypervariable V3 regions [26]. terms of plant growth promotion under salinity and drought PCR reactions were performed in a 50 Lfinalvolume stress it is essential to focus on the fraction of the culturable containing 1X buffer, 2.5 mM MgCl2,5%ofDMSO,0.12mM bacteria that is able to thrive under these specific conditions. of dNTPs mixture, 0.3 M of each primer, 1.5 U Taq poly- Therefore, the aims of this work were (i) the isolation of merase, and 10 ng of template, applying the following thermic ∘ ∘ halophilic/halotolerant bacteria from Salicornia rhizosphere protocol: 94 C for 4 min, followed by 10 cycles of 94 Cfor ∘ ∘ and bulk soils collected in hypersaline ecosystems in southern 0.5 min, 61 Cfor1min,and72C for 1 min; followed by ∘ ∘ ∘ Tunisia, (ii) the characterization of their resistance to abiotic further 20 cycles of 94 Cfor0.5min,56 Cfor1min,and72 C ∘ stresses and their plant growth promoting (PGP) potential, for 1 min; and a final extension at 72 Cfor7min.Presence and (iii) the description of taxonomic diversity of both the andlengthofPCRproductswereverifiedbyelectrophoresis halophilic/halotolerant culturable fraction and the whole in 1% w/v agarose gel prior to Denaturing Gradient Gel bacterial microbiome inhabiting Salicornia rhizosphere and Electrophoresis (DGGE) analysis. bulk soils. 2. Materials and Methods 2.3. Denaturing Gradient Gel Electrophoresis. PCR products (∼150 ng) were loaded in a 0.5 mm polyacrylamide gel 2.1. Site Description, Soil Sampling, and Soil Characterization. (7% (w/v) acrylamide-bisacrylamide, 37.5: 1) containing 40 ∘ ∘ The studied sites, named BDV4 (N 34 26 951 ;E09 54 102 ), to 60% urea-formamide denaturing gradient (100% corre- ∘ ∘ BDV11 (N 34 08 735 ;E0804 417 ), and BDV20 (N sponds to 7 M urea and 40% (vol/vol) formamide) according ∘ ∘ 33 57 252 ;E0824 508 ) corresponded, respectively, to to the method described by Muyzer et al. [26]. The gels were ∘ Sebkhet El Naouel, Chott El Gharsa, and Chott El Jerid and run for 15 h at 60 Cbyapplyingaconstantvoltageof90V were located in southern Tunisia. in 1X Tris-acetate-EDTA (TAE) buffer. After electrophoresis, Visual inspection of the sites identified Salicornia as the the gels were stained for 30 min in 1X TAE buffer containing only present plant. The plants were identified according to the 1X SYBR Green (Molecular Probes, Leiden, the Netherlands) plant morphology as S. strobilacea [23, 24], a widespread plant according to manufacturer’s instructions and rinsed twice for in southern Tunisia. 10 min with distilled water. Gels images were captured using Between the sites different conditions in respect of super- a Gel Doc 2000 apparatus (Bio-Rad, Milan, Italy). The band ficial salt