Potential for Plant Growth Promotion of Rhizobacteria Associated with Salicornia Growing in Tunisian Hypersaline Soils

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 248078 13 pageshttpdxdoiorg1011552013248078

Research ArticlePotential for Plant Growth Promotion ofRhizobacteria Associated with Salicornia Growing inTunisian Hypersaline Soils

Francesca Mapelli1 Ramona Marasco1 Eleonora Rolli1 Marta Barbato1 Hanene Cherif2

Amel Guesmi2 Imen Ouzari2 Daniele Daffonchio1 and Sara Borin1

1 DeFENS Department of Food Environment and Nutritional Sciences (DeFENS) University of Milan Via Celoria 220133 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 saraborinunimiit

Received 15 March 2013 Revised 30 April 2013 Accepted 3 May 2013

Academic Editor George Tsiamis

Copyright copy 2013 Francesca Mapelli et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Soil salinity and drought are among the environmental stresses that most severely affect plant growth and production aroundthe world In this study the rhizospheres of Salicornia plants and bulk soils were collected from Sebkhet and Chott hypersalineecosystems in Tunisia Depiction of bacterial microbiome composition by Denaturing Gradient Gel Electrophoresis unveiled theoccurrence of a high bacterial diversity associated with Salicornia root system A large collection of 475 halophilic and halotolerantbacteria was established from Salicornia rhizosphere and the surrounding bulk soil and the bacteria were characterized for theresistance to temperature osmotic and saline stresses and plant growth promotion (PGP) features Twenty Halomonas strainsshowed resistance to a wide set of abiotic stresses and were able to perform different PGP activities in vitro at 5 NaCl includingammonia and indole-3-acetic acid production phosphate solubilisation and potential nitrogen fixation By using a gfp-labelledstrain it was possible to demonstrate that Halomonas is capable of successfully colonising Salicornia roots in the laboratoryconditions Our results indicated that the culturable halophilichalotolerant bacteria inhabiting salty and arid ecosystems havea potential to contribute to promoting plant growth under the harsh salinity and drought conditionsThese halophilichalotolerantstrains could be exploited in biofertilizer formulates to sustain crop production in degraded and arid lands

1 Introduction

The influence of microbes on plant fitness has been rec-ognized both in conventional and extreme habitats wherethe ability of rhizobacteria to facilitate plant adaptation andpromote growth and productivity has been reported [1ndash6]Root-associated bacteria can promote plant growth by directand indirect mechanisms the former including nutrientfixation and solubilisation and phytohormones synthesisIndirect activities include biocontrol the ability to reduce oravoid the harmful effects of phytopathogens Both the hostplant and its associated microbiome gain an evolutionaryadvantage to survive under harsh conditions by establishingtight interplays

Among abiotic stresses soil salinity is one of the strongestfactors affecting plant growth and yield [7] Conditions ofhigh salt concentrations in the soil are very frequent in aridand semiarid regions on Earth where different halophyticspecies can be found Halophytes have been proposed askey players for saline soils reclamation [8] phytoremediationof hydrocarbon and heavy metals polluted saline soils [910] and forage and oil seed production [11 12] Salicornia(Chenopodiaceae) is a subcosmopolitan plant genus com-prising annual species strictly occurring in salty environ-ments and widespread in several countries including thoseof the Mediterranean basin Salicornia densely colonisesdifferent areas of southern Tunisia including Sebkhet andChott ecosystems dominated by extreme values of aridity

2 BioMed Research International

and soil salinity Intense evaporation rates render Sebkhet andChott as dry salt lakes which are inhospitable for most of theorganisms

The manipulation of natural resources to increase plantproductivity in lands traditionally considered unsuitable foragriculture is a challenging but necessary task in the lightof the increasing world population and the need for foodproduction [13] The efforts which aimed to the productionof salt-resistant crops include conventional breedingmarker-assisted selection and the creation of transgenic plants andare nowadays focusing also on the halophyte potential toguarantee a suitable food production in a salinized planet[14] Different works in the last years highlighted the impor-tance of plant growth promoting bacteria in facilitating salttolerance in plants devoted to food production [3 7 1516] and few reports emphasized the role of PGP bacteriaassociated with Salicornia spp [17ndash22] The investigation ofthe rhizobacterial community associated to plants naturallyadapted to cope with extreme saline conditions might lead toseveral knowledge outputs (i) the understanding of the plant-microbe interaction under saline conditions (ii) definitionof the mechanisms underlying plant growth with promotionunder the salinity stress and (iii) identification of bacterialstrains to design biological fertilizers exploitable for agricul-ture in arid and saline lands To achieve the best results interms of plant growth promotion under salinity and droughtstress it is essential to focus on the fraction of the culturablebacteria that is able to thrive under these specific conditionsTherefore the aims of this work were (i) the isolation ofhalophilichalotolerant bacteria from Salicornia rhizosphereand bulk soils collected in hypersaline ecosystems in southernTunisia (ii) the characterization of their resistance to abioticstresses and their plant growth promoting (PGP) potentialand (iii) the description of taxonomic diversity of both thehalophilichalotolerant culturable fraction and the wholebacterial microbiome inhabiting Salicornia rhizosphere andbulk soils

2 Materials and Methods

21 Site Description Soil Sampling and Soil CharacterizationThestudied sites namedBDV4 (N 34∘26101584095110158401015840 E 09∘54101584010210158401015840)BDV11 (N 34∘08101584073510158401015840 E 08∘04101584041710158401015840) and BDV20 (N33∘57101584025210158401015840 E 08∘24101584050810158401015840) corresponded respectively toSebkhet El Naouel Chott El Gharsa and Chott El Jerid andwere located in southern Tunisia

Visual inspection of the sites identified Salicornia as theonly present plantThe plants were identified according to theplantmorphology as S strobilacea [23 24] awidespread plantin southern Tunisia

Between the sites different conditions in respect of super-ficial salt crust presence (BDV11) or absence (BDV20) wereobserved (Table 1) Rhizospheric and bulk soils were sampledfrom triplicate specimens of Salicornia from sites BDV11and BDV20 In the site BDV4 different microenvironmentswere identified and a total of six Salicornia specimens werecollected In this site three sampled specimens were growingon salt crust covered soil (BDV4-S1 BDV4-S2 and BDV4-S3) and three sampled specimens were growing on a soil

plot where salt crusts were absent (BDV4-S4 BDV4-S5 andBDV4-S6) Replicates of bulk soils were also sampled fromthe site BDV4 (presence of salt crust BDV4-B1 absenceof salt crust BDV4-B4) Rhizospheric soil was defined assoil particles tightly adhering to roots (1ndash3mm) after gentlyshaking Bulk soil was collected as control about 2m farfrom any vegetation Rhizosphere and bulk soils will berespectively indicated in the text with the codes S and BSoil samples were collected using sterile spoons and storedin sterile bags at minus20∘C for molecular analyses and at 4∘C formicrobiological isolation Soil salinity was measured with ahand refractometer (Atago Tokyo Japan) after the extractionof pore water from approximately 2 g of soil

22 Metagenome Extraction and 16S rRNA AmplificationDNA was extracted from 05 g of soil using the protocolestablished by Schbereiter-Gurtner et al [25]

DNA was quantified using NanoDrop 1000 spectropho-tometer (Thermo Scientific Waltham MA USA)

Bacterial 16S rRNA gene fragments (sim550 bp) were PCRamplified using primers 907R (31015840-CCGTCAATTCCTTTG-AGTTT-51015840) and GC-357F (31015840-CCTACGGGAGGCAGCAG-51015840 with a 51015840-end GC-clamp) targeting a portion of the 16SrRNA gene that include the hypervariable V3 regions [26]PCR reactions were performed in a 50 120583L final volumecontaining 1X buffer 25mMMgCl

2 5 of DMSO 012mM

of dNTPs mixture 03 120583M of each primer 15 U Taq poly-merase and 10 ng of template applying the following thermicprotocol 94∘C for 4min followed by 10 cycles of 94∘C for05min 61∘C for 1min and 72∘C for 1min followed byfurther 20 cycles of 94∘C for 05min 56∘C for 1min and 72∘Cfor 1min and a final extension at 72∘C for 7min Presenceand length of PCR products were verified by electrophoresisin 1wv agarose gel prior to Denaturing Gradient GelElectrophoresis (DGGE) analysis

23 Denaturing Gradient Gel Electrophoresis PCR products(sim150 ng) were loaded in a 05mm polyacrylamide gel(7 (wv) acrylamide-bisacrylamide 375 1) containing 40to 60 urea-formamide denaturing gradient (100 corre-sponds to 7M urea and 40 (volvol) formamide) accordingto the method described by Muyzer et al [26] The gels wererun for 15 h at 60∘C by applying a constant voltage of 90Vin 1X Tris-acetate-EDTA (TAE) buffer After electrophoresisthe gels were stained for 30min in 1X TAE buffer containing1X SYBR Green (Molecular Probes Leiden the Netherlands)according tomanufacturerrsquos instructions and rinsed twice for10min with distilled water Gels images were captured usinga Gel Doc 2000 apparatus (Bio-Rad Milan Italy) The bandpatterns of DGGE gels were analysed using Image J software(available for free download at httprsbinfonihgovij) andMicrosoft Excel XLSTAT software (Addinsoft Inc NewYorkNY USA) as previously described [5] DGGE bands wereexcided from the gels with a sterile scalpel and eluted in 50 120583Lof sterileMilli-Qwater at 37∘C for 4 hours Subsequently 8120583Lof eluted DNA was reamplified by PCR using primers 357Fand 907R as described in the previous paragraph Positive

BioMed Research International 3

Table 1 Sample code location and characteristics of the Salicornia rhizospheres and bulk soils collected in Tunisia and analysed in the presentstudy

Sample code Soil fraction Site Coordinates FeatureBDV4-S123 Rhizosphere Sebkhet en Naouel N 34∘26101584095110158401015840 E 09∘54101584010210158401015840 soil covered by salt crustBDV4-S456 Rhizosphere Sebkhet en Naouel N 34∘26101584095110158401015840 E 09∘54101584010210158401015840 soil

BDV11-S123 Rhizosphere Chott El Gharsa N 34∘08101584073510158401015840 E 08∘04101584041710158401015840 soil covered by salt crust(173 plusmn 13 of salinity)

BDV20-S123 Rhizosphere Chott El Jerid N 33∘57101584025210158401015840 E 08∘24101584050810158401015840 soilBDV4-B1-123 Bulk soil Sebkhet en Naouel N 34∘26101584095110158401015840 E 09∘54101584010210158401015840 soil covered by salt crustBDV4-B4-123 Bulk soil Sebkhet en Naouel N 34∘26101584095110158401015840 E 09∘54101584010210158401015840 soil

BDV11-B123 Bulk soil Chott El Gharsa N 34∘08101584073510158401015840 E 08∘04101584041710158401015840 soil covered by salt crust(191 plusmn 04 of salinity)

BDV20-B123 Bulk soil Chott El Jerid N 33∘57101584025210158401015840 E 08∘24101584050810158401015840 soil

amplifications were partially sequenced by Macrogen IncKorea (httpwwwmacrogencom) using the primer 357F

24 Bacteria Isolation Rhizospheric and bulk soil triplicateswere pooled prior to bacteria isolation except in the case ofthe specimens BDV4-S4 BDV4-S5 and BDV4-S6 collectedat site BDV4 in absence of surface salt crust and processedseparately Thus we compared the structure of culturablehalotoleranthalophilic bacteria among the different studysites and additionally the taxonomic diversity within thesame site was evaluated by comparing the replicates Todetermine the bacterial cell number 1 g of rhizospheric andbulk soils collected at the different sites was shaken with9mL of sterile saline solution (09 NaCl) The suspensionswere serially diluted and plated in triplicate on solidifiedR2A medium (Oxoid) enriched with 10 and 15 NaCl After1-week incubation at 30∘C the colony-forming unit (cfu)per gram was determined and for each sample a numberof colonies comprised between 8 and 42 per medium wererandomly selected The bacterial isolates were stored as 25glycerol stocks at minus80∘C

25 Genotypic Characterization and Identification DNAextraction was performed on each isolated strain The bacte-ria collection has been dereplicated through the applicationof 16S-23S rRNA Intergenic Transcribed Spacer-PCR (ITS-PCR) using ITS-F (31015840-GTCGTAACAAGGTAGCCGTA-51015840)and ITS-R (31015840-CTACGGCTACCTTGTTACGA-51015840) primersas previously described [5 27] PCR amplification wasperformed in 25120583L reaction containing 1X buffer 15mMMgCl

2 012mM of dNTPs mixture 03 120583M of each primer

1 U Taq polymerase and 10 ng of template applying thefollowing thermic protocol 94∘C for 4min followed by 35cycles of 94∘C for 05min 55∘C for 1min and 72∘C for2min and a final extension at 72∘C for 10min The ITS-PCR products were run on 2 agarose gels and stainedwith ethidium bromide Gel images were captured usinga Gel Doc 2000 apparatus (Bio-Rad Milan Italy) andITS-fingerprinting profiles were visually analysed to clus-ter together the bacterial isolates showing the same bandpattern From each cluster at least a representative strainhas been selected for subsequent PGP characterization and

genotypic identification through 16S rRNA gene sequenc-ing 16S rRNA amplification was performed by using theuniversal primers 27F (31015840-AGAGTTTGATCMTGGCTCAG-51015840) and 1492R (31015840-CTACGGCTACCTTGTTACGA-51015840) [28]and applying the same protocol of ITS-PCR Partial 16SrRNA sequences were obtained from Macrogen Inc Korea(httpwwwmacrogencom)

26 Nucleotide Sequence Analyses and Accession NumbersNucleotide sequences were edited in Chromas Lite 201(httpwwwtechnelysiumcomau) and subjected to BLASTsearch (httpblastncbinlmnihgovBlastcgi) The partial16S rRNA gene sequences obtained from the bacterial isolatesand the excised DGGE bands have been deposited in theEMBL respectively under accession numbers HF678717ndashHF678862 and HF678127ndashHF678194

27 Resistance to Abiotic Stresses Resistance to salt stress wasassessed by growing the isolates at 30∘C inR2A supplementedby different sodium chloride concentrations ranging from0 to 20wv The ability to grow under osmotic stress wastested at 30∘C by adding 5ndash20 of polyethylene glycol (PEG)to R2A broth medium Finally the capability to growth in awide range of temperatures was verified by incubating theR2A plates at 4∘ 42∘ and 50∘C A control consisting in asterile plate or tube was also run parallel to each experiment

28 In Vitro Screening of Plant Growth Promoting ActivitiesEach isolate was grown as pure culture to evaluate its PGPfeatures in suitable media enriched with 5wv NaCl Onlytwo isolates were not able to grow at 5 NaCl and theirPGP activities were tested at 10 NaCl The production ofindole-3-acetic acid was detected by the method describedby Bric et al [29] The ability to solubilise insoluble phos-phate compounds was estimated according to Ahmad etal [30] The ammonia synthesis assay was performed asrecommended by Cappuccino and Sherman [31] Proteaseactivitywas determined fromclearing zones in skimmedmilkagar according to Nielsen and Soslashrensen [32] Atmosphericnitrogen fixation ability and ACC-deaminase activity weredetermined by the method of Penrose and Glick [33] nifH

4 BioMed Research International

gene detection has been performed by PCR test usingthe primer sets PolF (31015840-TGCGAYCCSAARGCBGACTC-51015840)and PolR (31015840-ATSGCCATCATYTCRCCGGA-51015840) [34] PCRamplification was performed in 25120583L reaction containing 1Xbuffer 15mM MgCl

2 012mM of dNTPs mixture 03 120583M

of each primer 1 U Taq polymerase and 10 ng of templateapplying the following thermic protocol 94∘C for 4minfollowed by 35 cycles of 94∘C for 05min 55∘C for 1min and72∘C for 2min and a final extension at 72∘C for 10min

29 Chromosomal gfp-Tagging of Halotolerant HalomonasStrains by Conjugation Procedure To stably transform strainsaffiliated to the genus Halomonas we adopted the methodbased on mini-Tn7 transposon system [35] Briefly themobilisation of the gfp-harbouring fragment was achieved bya four-parental conjugation formed by a cellular suspensionof 1010 cells of the strain to be transformed and 109 cellsfor E coli strains carrying helper delivery and mobilizationplasmids [35] To select for gfp-transformed cells after themating the cellular suspension was plated in R2A mediumsupplemented with 10 NaCl and the required antibioticsThe gfp-labelling procedure was successful for a strain of Helongata as visualized by fluorescence microscopy

210 In Vitro Bacterial Rhizocompetence Test To evaluategfp-labelled strain ability to adhere and potentially coloniseplant root system an in vitro assay was performed on twomodel plants Arabidopsis thaliana and Salicornia plantletscollected in marine dune ecosystems in south Italy Afteran overnight growth in liquid selective medium bacterialcell concentration was microscopically evaluated and a 108cellmL suspension was prepared Salicornia plant roots weredipped in MS salt half strength medium (SIGMA Italy)supplemented with 2 NaCl and the prepared bacterialsuspension For Arabidopsis rhizocompetence test NaCladdition was avoided since this plant is extremely salt stress-sensitive After an overnight incubation (sim16 h) plant rootswere gently washed to remove no- or weakly-bound bacterialcells and observed under a confocal laser scanning micro-scope (Leica TCSNT) Images were acquired using LeicaConfocal Software and analysed by using the MBF ImageJsoftware

3 Results and Discussion

31 DGGE Analysis of the Bacterial Microbiome InhabitingSalicornia Rhizosphere and Surrounding Bulk Soil The intro-duction of fingerprint-based analyses [26 36 37] turnedinto the application of cultivation-independent techniques asroutine tools to depict the overallmicrobiome composition inenvironmental samples and to infer which factors influencethe abundance and distribution of specific microbial taxaHere DGGE analysis of 16S rRNA gene was applied to pro-vide a snapshot of both culturable and unculturable bacterialassemblages in Salicornia rhizosphere and the surroundingbulk soil not affected by the plant Bulk soil samples fromall sites were analysed in triplicate except in BDV4-B4sites where only duplicate samples were analysed due to the

failing of PCR-DGGE amplification Although the bulk soilscollected fromTunisian Sebkhet andChottwere characterizedby extreme dryness and salinity values DGGE band profileshighlighted that a rich and diverse bacterial microbiome waspresent in all the samples (Figure 1(a) left panel) PrincipalComponent Analysis (PCA) performed on the line plotsderived from DGGE band profiles (Figure 1(b) left panel)indicated that bulk soils clustered according to the site ofprovenience On axis 1 describing the 44 of the samplessimilarity bulk soil samples were distributed according tothe presence (stations BDV4-B1 and BDV11-B) or absence(stations BDV20-B and BDV4-B4) of salt crusts coveringthe soil surface Salinity is known as one of the strongestabiotic factors influencing the assemblages of a huge varietyof bacterial populations sheltered by the soil [38]The factorsshaping the composition of the bacterial community includealso biotic interactions and the role of root exudates inthe selection of a peculiar microbiome is well known [39]The DGGE pattern obtained from Salicornia rhizospheresamples was different from those observed in bulk soils(Figure 1(a)) According to DGGE fingerprints the bacterialcommunities of the rhizospheric soil triplicates collected atboth sites BDV20 and BDV11 clustered together (Figure 1(a)right panel) Similarly the rhizospheres collected from thesalt crust covered soil at site BDV4 (BDV4-S1 2 and 3)showed a high level of homogeneity A high number ofDGGE bands were observed in all the rhizospheres with theexclusion of BDV4-S6 sample probably affected by biasesin PCR amplification Overall the rhizosphere of Salicorniawas proved to be a habitat characterized by a highly richbacterial community Principal Component Analysis of theDGGE patterns (Figure 1(b) right panel) indicated exceptfor rhizosphere samples BDV4-S5 and BDV4-S1 a highersimilarity among the rhizospheres collected from differentstations than among the bulk soils suggesting that therhizosphere acts as a selection factor that tend to uniformbacterial diversity independent from the soil type PrincipalComponent Analysis (Figure 1(b) right panel) pointed outthe overall similarity of the bacterial communities hostedby the Salicornia rhizospheres collected in different sitesThe even structure of the rhizosphere bacterial communityconfirmed the importance of plant inputs in the selectionof specific bacterial taxa associated with the roots a well-known phenomenon generally reported as ldquorhizosphereeffectrdquo Despite that the occurrence of a degree of variabilitywithin the bacterial microbiome associated with Salicorniaspecimens collected in different microenvironments withinsite BDV4 (related to presence and absence of salt crusts onsoil surface) was perceived by DGGE analysis This resultconfirmed that besides the selection driven by the plantalso the environmental parameters play a role in shaping therhizosphere bacterial microbiome

According to DGGE band sequencing analyses theprevalent taxonomic groups associated with Salicornia rootsand bulk soils were Alpha- Beta- and Gammaproteobac-teria Bacilli and Actinobacteria (Figure 1(c)) as previouslyobserved in the bacterial communities associated to dif-ferent plant species (Capsicum annuum) growing in desertareas under water stress condition [5] In addition two

BioMed Research International 5

BDV

20-B

1

BDV

20-B

2

BDV

20-B

3

BDV

11-B

1

BDV

11-B

2

BDV

11-B

3

BDV

4-B1

-1

BDV

4-B1

-2

BDV

4-B1

-3

BDV

4-B4

-1

BDV

4-B4

-2

BDV

20-S

1

BDV

20-S

2

BDV

20-S

3

BDV

11-S

1

BDV

11-S

2

BDV

11-S

3

BDV

4-S1

BDV

4-S2

BDV

4-S3

BDV

4-S4

BDV

4-S5

BDV

4-S6

13

16 15

25

6263

27

3

5

60

22

8

95051

49

42

444531

58

333435

3637

40

66

56

54

71

70

171

6667

68

74

75

79

78

178179

105106

838485

89

88

97

110

177

95

96

99

98

107

113

174

162

163

142

154

169

138

115

130

(a)

minus30 minus20 minus10 0 10 20minus20

minus10

0

10

20

F1 (4361)

F2 (2

276

)

BDV4-B4-2

BDV4-B4-1BDV4-B1-2

BDV4-B1-3

BDV4-B1-1

BDV20-B3BDV20-B2

BDV20-B1

BDV11-B3

BDV11-B2

BDV11-B1

minus1000 minus500 0 500 1000 1500minus1000

minus500

0

500

1000

F1 (5285)

F2 (1

34

) BDV4-S4BDV20-S1

BDV4-S5

BDV20-S2BDV11-S3

BDV11-S1BDV11-S2

BDV4-S5BDV20-S3

BDV4-S3

BDV4-S2BDV4-S1

(b)

0

5

10

15

20

BDV4-B1 BDV4-B4 BDV11-B BDV20-B

Num

ber o

f ba

nds

0

2

4

6

8

10

12

BDV4-S4-5-6 BDV4-S1-2-3 BDV11-S1-2-3 BDV20-S1-2-3

Num

ber o

f ban

ds

RhizobialesFlavobacterialesRhodobacteralesSphingobacterialesSphingomonadalesActinomycetales

BurkholderialesUnc ActinobacteriaLegionellalesUnc BacteriaBacillales

(c)

Figure 1 DGGE analysis performed on the bulk (left panel) and rhizospheric (right panel) soil bacterial community (a) In the left panelDGGE patterns of the bulk soils collected at sites BDV20 BDV11 and BDV4 In the right panel DGGE patterns of the rhizospheric soilscollected at sites BDV20 BDV11 and BDV4The numbers represented the three analysed replicates (b) Principal Component Analysis basedon the DGGE profiles of the bacterial community inhabiting bulk (left) and rhizospheric (right) soil associated with Salicornia specimens(c) Taxonomic identification of bacterial 16S rRNA sequences excised from DGGE bands cut from rhizospheric and bulk soil profiles

6 BioMed Research International

BDV

4-S1

BDV

4-S4

BDV

4-S5

BDV

4-S6

BDV

11-B

BDV

11-S

1

BDV

20-S

1

R2A 10R2A 15

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

1E+11

Soil

(cfu

g)

Figure 2 Evaluation of the halophilichalotolerant culturable bac-teria number of Salicornia rhizosphere and bulk soils Microbial cellnumber is reported as colony-forming unit (cfu) per gram of freshsoil Dark grey bars represent the cfu per gram of fresh soil detectedon R2A medium enriched with 10 NaCl Light grey bars representthe cfu per gram of fresh soil detected on R2A medium enrichedwith 15 NaCl

different orders belonging to the Bacteroidetes phylumwere retrieved namely Sphingobacteriales and Flavobac-teriales the latter exclusively present in the bulk soil(Figure 1(c)) Both in rhizospheric and bulk soils Beta- andGammaproteobacteria and Bacilli were represented by onlyone taxonomic order while Alphaproteobacteria composi-tion differed in the two soil fractions Alphaproteobacteriain rhizospheric soils were represented exclusively by Rhi-zobiales spp while bulk soils were colonised by a morediverse bacterial community including in addition to Rhi-zobiales the orders Sphingomonadales and Rhodobacterales

32 Bacteria Isolation and Identification Viable halotolerantbacteria were cultured on oligotrophic medium from all thecollected rhizospheres and from the bulk soil sampled atBDV11 site Culturable bacteria abundance (Figure 2) wasconsiderably variable between the different sites and thenumber of colony-forming units (cfu) ranged between 9 times104 and 16 times 1010 per gram of fresh soil Similar counts wereobserved within the sites on the samemedium supplementedwith 10 and 15 of sodium chloride This result indicatesthat most of the halotolerant culturable bacteria are ableto cope with the higher value of salt content close to thesalinity measured in the soil pore water of BDV11 stationHalotolerant bacteria abundance in the BDV11-B bulk soilwas in accordance with values previously reported in similarenvironments [40] The interaction with the plant and thepresence of root exudates could be responsible for the higherabundance of halotoleranthalophilic bacteria detected inthe rhizosphere (Figure 2) as compared to the bulk soils inBDV11 site These results are in agreement with the ldquorhizo-sphere effectrdquo described by several authors in conventionaland extreme environments [5 39] To our knowledge this

work is nevertheless the first report about the quantifica-tion of halotolerant microbes in root-associated extremelysaline soils enlarging the concept of the rhizosphere effectto specific bacterial groups highly adapted to the hostileenvironmental conditions

A large collection of 475 isolates representing the halotol-erant culturable fraction of the bacterial diversity associatedwith Salicornia specimens and bulk soil of Sebkhet andChott ecosystems was established In the case of BDV4-S6only eight colonies growing in a medium containing 15NaCl were isolated whilst for the rest of the samples anumber of colonies comprising between 22 and 42 were ran-domly picked for both the salt enrichment conditions ITS-PCR fingerprinting was applied to dereplicate the bacteriacollection allowing the identification of 136 clusters corre-sponding to different ITS profiles and representing differentspeciessubspecies From each haplotype at least one strainwas arbitrarily selected for 16S rRNA partial gene sequencingand for the phenotypic tests The taxonomic identification ofthe bacteria showed the prevalence of the Halomonas genusamong the strains isolated at 15 NaCl (Figure 3) Excludingsamples BDV4-S5 and BDV4-S6 where bacteria belonging tothe genus Nesterenkonia were also retrieved the strains iso-lated from both bulk and rhizosphere soils on medium con-taining 15NaClwere represented exclusively byHalomonasHence the subcollection obtained on 15 sodium chloridesupplementedmediumwas characterized by high dominanceand low values of the Shannon diversity index (Table 2)Similarly bacteria isolated on 10 NaCl containing mediumfrom BDV11-B and BDV11-S1 belonged exclusively to theHalomonas genus (Figure 3) This genus together with thegenus Chromohalobacter represented the totality of BDV20-S1 halophilic culturable community (Figure 3) The preva-lence of the Halomonas genus was demonstrated withinthe halotolerant root-associated bacteria collection whereHalomonas elongata along with the species H eurihalina Hsinaiensis H halmophila H ilicicola H indalina H vari-abilis H xinjiangensis and H taeheungii where retrievedThe isolation of Halomonas sp from the roots of Salicorniabrachiata was recently reported [19] Besides harbouringhigher salt tolerant bacterial counts Salicornia rhizospherewas richer than bulk soils also in terms of biodiversitysince all the bacteria isolated from BDV11-B belonged tothe specie Halomonas elongata Overall the strains isolatedat 10 NaCl from rhizosphere soil of site BDV4 displayeda higher biodiversity at the genus level (Figure 3) that isreflected by low dominance values and high values of theShannon diversity index (Table 2) BDV4 rhizospheric soilscharacterized by lower abundance of culturable halotoler-anthalophilic bacteria compared to those collected at sitesBDV11 and BDV20 (Figure 2) hosted a more diverse halo-tolerant bacterial community comprising different generathat were previously reported in other hypersaline environ-ments [41] Strains belonging to the Chromohalobacter genuswere isolated at 10 NaCl from BDV4-S6 and BDV20-S1This genus along with Halomonas represents an importantmember of the family Halomonadaceae a taxonomic groupwithin the Gammaproteobacteria typical of hypersaline envi-ronments whose taxonomy is still under revision [42] A

BioMed Research International 7

Table2

Diversityindicesof

halotoleranthalop

hilic

bacteria

collection

Thecalculationbasedon

thegenera

distrib

utionin

thedifferent

analysed

rhizosph

eric

androot-fr

eesoilsTh

epercentage

indicatesinbracketsrefersto

theN

aClcon

centratio

nused

intheisolatio

nmedium

BDV4-S1

(10

)BD

V4-S1

(15

)BD

V4-S4

(10

)BD

V4-S4

(15

)BD

V4-S5

(10

)BD

V4-S5

(15

)BD

V4-S6

(10

)BD

V4-S6

(15

)BD

V20-S1

(10

)BD

V20-S1

(15

)BD

V11-B

(10

)BD

V11-B

(15

)BD

V11-S1

(10

)BD

V11-S1

(15

)Genera

41

41

42

52

21

11

11

Individu

als28

38

32

32

24

40

22

842

42

42

42

42

42

Dom

inance04668

103203

10538208613034710781308673

11

11

1

Shanno

n09887

01245

0088240266412540376802573

00

00

0

Evenness06719

108682

10604206526070060728806467

11

11

1

8 BioMed Research International

05

1015202530354045

Num

ber o

f iso

late

s

BDV

4-S1

-10

BDV

4-S4

-10

BDV

4-S5

-10

BDV

4-S6

-10

BDV

20-S

1-10

BDV

11-B

-10

BDV

11-S

1-10

BDV

4-S1

-15

BDV

4-S4

-15

BDV

4-S5

-15

BDV

4-S6

-15

BDV

20-S

1-15

BDV

11-B

-15

BDV

11-S

1-15

Chromohalobacter

Halobacillus

Halomonas

KushneriaMarinococcus

Nesterenkonia

Oceanobacillus

Virgibacillus

Figure 3 Taxonomic composition of the halophilichalotolerantfraction of culturable bacteria associatedwith Salicornia rhizosphereand bulk soils shown as genera distribution The numbers 10 and 15in the sample name indicate the percentage of NaCl supplementedto the medium during isolation procedures

third genus Kushneria of the family Halomonadaceae wasisolated from the BDV4-S4 rhizosphereThe additional root-associated halotolerant bacteria belonged to the classes Bacilli(Halobacillus trueperiMarinococcus halophilusOceanobacil-lus picturae and Virgibacillus olivae) and Actinobacteria thelatter being represented exclusively by the speciesNesterenko-nia halobia Both Oceanobacillus picturae and Nesterenkoniahalobia were previously isolated from a different salineecosystem namely mangrove sediments [43 44] where Opicturea was described for the first time as a phosphate-solubilising bacterium able to promote mangrove seedling

Taxonomic analyses on the bacteria isolated from therhizosphere of plants growing in different niches of thesame site (BDV4-S4 BDV4-S5 and BDV4-S6) permittedto assess the occurrence of intrasite environmental selectiveforces shaping the composition of the culturable halophiliccommunity as it was already shown for the total bacterialcommunity by DGGE-fingerprinting results Even thoughthe enrichment on 15 NaCl resulted in an even taxonomicdistribution of the isolates a certain degree of variabilitycould be observed in the bacteria isolated at 10 NaCl(Figure 3) suggesting that microvariations within the sitemay influence the prevalence of different bacterial popu-lations in the culturable halotolerant fraction This resultstrengthens the diversity pattern described by DGGE on thetotal bacterial microbiome inhabiting the Salicornia rhizo-spheric soils collected at site BDV4 (Figure 1 right panel)indicating a partial overcoming of environmental factors onthe rhizosphere effect imposed by the host plant

33 Resistance to Abiotic Stresses Aiming to identify themostsuitable rhizobacteria to design a biofertilizer for sustainingplant growth in saline and arid soils the ability of the isolatedbacteria to cope with different abiotic stresses typical ofarid lands was tested on 164 bacterial strains belonging to

0102030405060708090

100

0NaCl

5NaCl

10NaCl

15NaCl

20NaCl

5PEG

10PEG

20PEG

Isol

ates

()

4∘C 42∘C 50∘C

(a)

0102030405060708090

100

Isol

ates

()

IAA P-sol N-fix NH3 Prot ACC

(b)

Figure 4 Spread of abiotic resistance and plant growth promotingtraits among the halophilichalotolerant bacteria isolated fromSalicornia rhizosphere and bulk soils (a) Abiotic stresses resistancePEG polyethylene glycol (b) Plant growth promotion features IAAindole-3-acetic acid production P-sol phosphate solubilization N-fix putative nitrogen fixation ability NH

3

ammonia productionProt Protease activity ACC 1-aminocyclopropane-1-carboxylatedeaminase

the 136 ITS groups identified by collection dereplicationIn particular the bacteria collection was screened for thecapability (i) to grow at extreme temperature values (ii)to thrive in presence of different salt concentrations and(iii) in conditions of low water availability The strains ableto grow at 42∘C represented 93 of the bacteria collection(Figure 4(a)) whereas only 13 of the isolates survived athigher temperature (50∘C) The ability to flourish at lowtemperature (4∘C) was observed for 71 of the total isolatesand twenty strains were able to grow in a large temperatureinterval between 4 and 50∘C In arid and saline soils thevegetation is generally sparse a factor that contributes to thestrong temperature fluctuations affecting the soil Hence theability of the plant associated microbes to grow in a largetemperature range and to survive at temperature fluctuationsis useful to efficiently colonise barren and extreme deserthabitats

All the isolates were isolated in the presence of 10 and 15of sodium chloride in the growing medium The majority ofthe isolates corresponding respectively to 99 and 92 ofthe bacteria collection grew at lower (5) or higher (20)salinity (Figure 4(a)) A significant fraction of the collection(74) was constituted by halophiles unable to grow in theabsence of NaCl in the medium (Figure 4(a))

A high percentage (90) of the isolated bacteria wasable to grow in presence of 5 polyethylene Glycol (PEG)(Figure 4(a)) a molecule which induces a decrease of thewater potential when added to the cultivation media [45]A decline in the number of isolates that positively grew atincreasing PEG concentration was observed nevertheless the

BioMed Research International 9

percentage of bacteria able to grow at 10 and 20 of PEGwerenoteworthy and corresponded respectively to 87 and 81 ofthe bacteria collection

Tolerance to abiotic stresses was widespread within thebacteria collection and represented a common trait evenin phylogenetic unrelated strains as expected since manyof the retrieved species were previously isolated from salineand hypersaline habitats as in the case of Virgibacillus spp[46 47] Halomonas sinaiensis [48] and Kushneria andHalomonas spp [49 50]

In vitro tests showed that twenty Halomonas strainswere particularly resistant to extreme values of differentabiotic factors These isolates belonging to the species Helongata and H sinaiensis were able to actively grow (i) onR2A medium containing a percentage of sodium chloridecomprised between 5 and 20 (ii) in the presence of 5 10and 20 of PEG in the medium and (iii) when incubated ina wide range of temperature (from 4 to 50∘C)

The resistance of the isolates to the extreme physical-chemical parameters of Tunisian Sebkhet and Chott ecosys-tems is a prerequisite to select efficient PGP bacteria able tosustain plant growth since the effectiveness of a microbialconsortium strictly depends on its competitive root coloni-sation [51 52] a reason that explains why the use of PGPbacteria isolated from different soil and climate conditionscan be a largely unsuccessfully strategy in arid and salinelands [53 54]

34 Plant Growth Promotion Test The PGP activities of164 isolates belonging to the 136 ITS-PCR clusters andrepresenting the whole taxonomic diversity of the establishedbacteria collection were tested in vitro by using specificmedia supplemented by 5 sodium chloride

One of the strategies adopted by PGP bacteria to induceplant growth is the influence on the plant hormonal balance96 of the isolates showed the ability to produce indole-3-acetic acid (IAA) (Figure 4(b)) one of the main planthormones of the auxin family This trait was shared by allthe genera retrieved from the analysed rhizospheric and bulksoils while it was not detected in the isolatedChromohalobac-ter marismortui and C salexigens strains Few strains unableto produce IAA belong to the speciesOceanobacillus picturaeand Halomonas halophila characterized by an uneven dis-tribution of this PGP feature within their ITS clusters Thecapability to modulate the plant stress level by providingindole-3-acetic acid (IAA) a molecule involved in lateralroots development was previously reported for halotolerantbacteria isolated from coastal soils [55] halophyte roots inArgentina [56] and rhizosphere of C annum growing indesert areas [5] In addition the recent study by Tiwari et al[16] demonstrated that inoculation of wheat withHalomonassp the most abundant genus in our strains collectionresulted in higher content of IAA in the rhizosphere of thetreated plants than control experiment

Rhizobacteria can also positively influence the healthstatus of the host plant by reducing the concentration ofstress signaling molecule such as 1-aminocyclopropane-1-carboxylate a precursor of ethylene Only three strains out ofthe collection belonging to the speciesHalomonas taeheungii

and Halomonas xinjiangensis displayed ACC-deaminaseactivity in presence of 5 NaCl (Figure 4(b)) ACC-deaminase activity in the genus Halomonas was recentlyreported in the ambit of the investigation of PGP featuresof halophilic bacteria isolated from halophytes includingthe species Salicornia brachiata [22 55] Nonetheless thelow percentage (2) of ACC-deaminase activity amongthe collection established in this work is in agreement withprevious studies reporting the detection of ACC-deaminaseactivity only for a minor fraction of bacteria isolated fromthe rhizosphere of wheat growing in salinized soil [6 16]

Direct mechanisms of plant growth promotion includethose metabolisms that by supplying nutrients to the plantenhance its fitness The established halotoleranthalophilebacteria collection was analysed for the capability to solu-bilise phosphate fix nitrogen and produce ammonia Thephosphate solubilisation activity was present in 65 of thewhole collection (Figure 4(b)) including all the genera exceptfor Kushneria The potential activity of nitrogen fixation hasbeen phenotypically tested by the strain capability to growin nitrogen-free medium and confirmed by molecular inves-tigation by PCR amplification of the nifH gene codifyingfor a subunit of the nitrogenase enzyme Six percent of theanalysed bacterial strains were positive to both the testsshowing the putative ability to fix nitrogen (Figure 4(b))Putative nitrogen fixation activity was detected in only aminor fraction of the ITS clusters belonging to the speciesHalomonas elongata H eurihalina H indalina Kushneriamarisflavi andChromohalobacter canadensis Ammonia pro-duction was also a common PGP trait shown by 93 ofthe isolates (Figure 4(b)) All the bacteria genera present instrain collection were positive to the ammonia productionassay thus potentially contributing to plant nitrogen nutri-tion The inability to produce ammonia did not show anyspecies-related patternThewidespread ability to increase theconcentration of bioavailable nutrients in the isolate collec-tion from Salicornia rhizosphere suggested the contributionof these halotolerant and halophilic bacteria to the plantnutrient balance These direct PGP features were generallysimultaneously present in the same strain possibly actingin a synergic manner to directly promote plant growth aspreviously reported [30]

Besides direct PGP activity several representatives of allthe taxonomic classes retrieved in our collection (11) alsodisplayed in vitro protease activity a result that indicatedtheir possible role as biocontrol agents The bacterial iso-lates displaying protease activity comprised bacterial strainsof the genera Chromohalobacter Halomonas KushneriaMarinococcus Nesterenkonia and Virgibacillus

The results about the investigation of the PGP traitsoccurrence among the bacteria collection established fromthe Salicornia rhizospheric and bulk soils are in generalagreement with the observations reported by other studiesrealized on halophyte [22 55 56] and crop plant growingunder saline conditions [5 6 15 16]

35 In Vitro Colonisation of Salicornia Root System Besidesperforming in vitro activities involved in biostimulation

10 BioMed Research International

(a)

lowast

lowast

(b)

lowast

lowast

(c)

Figure 5 Representative images of gfp-taggedHalomonas elongata strain on Salicornia root acquired through BP53030GFP filter (excitationat 488 nm) (a) Fluorescence image showing gfp-H elongata cells and microcolonies (b) Bright field image of (a) showing Salicornia rootsurface (open arrow) and root hairs (arrow) (c) Overlapping of images (a) and (b) showing the colonisation of Salicornia root surface (openarrow in the upper right of the panel) and root hairs (arrow on the right side of the panel) by the gfp-tagged H elongata strain Asterisksindicated in the bright field images (a and b) show the biofilm matrix associated with the root surface The scale bars of the images in thefigure correspond to 10 120583m

biocontrol or biofertilization to play an effective role inplant growth promotion a bacterial strain should be able tocolonise the plant root system The potential ability of PGPisolates to efficiently colonise plant root system was testedby performing an adhesion assay exploiting a gfp-taggedPGP bacterium [57] The adhesion test was performed ona nonhalophyte model plant Arabidopsis thaliana alreadyused to study plant-microbe interactions [5 58 59] andon a wild Salicornia collected in southern Italy Differentrhizospheric bacterial strains belonging to the Halomonasgenus were selected based on their promising multiple PGPactivities in vitro and the ability to cope with several abioticstresses as candidate for the chromosomal gfp-tagging Helongata strain BDV11S17Awas successfully transformedwiththe gene encoding the Green Fluorescent Protein (gfp) thatwas stably inserted in the bacteriumgenomeThe gfp-labelledH elongata BDV11S17A was used to track bacterial adhesionon Arabidopsis and Salicornia roots in vitro by exposing theroots to a gfp-labelled bacterial suspension for 16 hoursThe gfp-tagged strain was unable to colonise Arabidopsisroot system and despite several attempts and the analysisof different root specimens only few cells were observedat the fluorescence microscope On the contrary confocalanalysis of Salicornia roots showed an extensive colonisationby gfp-labelled strain (Figure 5)H elongata BDV11S17A-gfppreviously shown to be able to grow under different stresses(high and low temperatures high saline concentrations andwater stress) and to perform PGP activities in vitro showed agood rhizocompetence efficiently colonizing Salicornia rootsurface and root hairs Such features make the strain apotential candidate for in vivo PGP experiments

4 Conclusions

DGGE fingerprinting on the total bacterial microbiomecolonising the bulk soils showed that the presenceabsence

of salt crusts on the soil surface was a driving force involvedin shaping the structure of the hypersaline soil dwellingbacterial community The same approach demonstrated thatSalicornia selected similar bacterial communities in the rhi-zosphere independently from the site of sampling Notablyrhizosphere associated bacterial communities differed fromthat colonizing the root-free soil Overall DGGE fingerprint-ing indicated that a peculiar bacterial microbiome is stablyassociated with Salicornia roots possibly having a role inpromoting plant growth and stress tolerance

The establishment of a large collection of halophilicand halotolerant bacterial strains and their identificationwidened the knowledge on the rhizocompetent bacterialcommunity associated with halophytes in saline and aridsoils Furthermore the isolation from the halophilic plantSalicornia of a large collection of bacteria which toleratetemperature saline and osmotic stresses and also showed invitro the ability at medium-high salinity value (5 NaCl) to(i) positively influence the nutrients and hormonal balanceand (ii) putatively express biocontrol activity as indicatedby the protease activity test is a novelty presented in thisstudy Furthermore the gfp-labelled PGP Halomonas elon-gata strain isolated from rhizospheric soil showed the abilityto massively adhere on Salicornia roots in vitro demon-strating the suitability of halophilic plants rhizobacteria toset up effective PGP inocula The great potential of PGPhalophilic and halotolerant bacteria should be carefully takeninto account to satisfy the increased need of food productionin the frame of a raising world population and ongoingclimate changes The present work contributes to expandthe current knowledge on PGP bacteria presenting a widebacterial strain collection that could be exploited to set upspecifically designed microbial consortia able to enhanceplant growth and productivity in soils impacted by salt anddrought stresses

BioMed Research International 11

Acknowledgments

Theauthors would like to thankDr Umberto Fascio ((CIMA)Centro Interdipartimentale di Microscopia avanzata of theUniversity of Milan) for technical support at the confocalmicroscope and Raffaella Tassoni for technical support inperforming the PGP tests The authors are grateful to DrLotte Lambertsen for the kind gifts of the E coli strainsfor gfp-labelling of the bacteria This work was financiallysupported by the European Union in the ambit of ProjectBIODESERT (European Communityrsquos Seventh FrameworkProgramme CSA-SA REGPOT-2008-2 under Grant agree-ment no 245746) F Mapelli and E Rolli were supportedby Universita degli Studi di Milano DeFENS EuropeanSocial Found (FSE) and Regione Lombardia (Contract ldquoDoteRicercardquo) R Marasco was supported by a fellowship in theambit of BIOGESTECA Project (n∘ 15083RCC ldquoFondo perla promozione di accordi istituzionalirdquo)

References

[1] A Balloi E Rolli R Marasco et al ldquoThe role of microorgan-isms in bioremediation and phytoremediation of polluted andstressed soilsrdquo Agrochimica vol 54 no 6 pp 353ndash369 2010

[2] L E de-Bashan J P Hernandez Y Bashan and R M MaierldquoBacillus pumilus ES4 candidate plant growth-promoting bac-terium to enhance establishment of plants in mine tailingsrdquoEnvironmental and Experimental Botany vol 69 no 3 pp 343ndash352 2010

[3] D Egamberdieva F Kamilova S Validov L Gafurova ZKucharova and B Lugtenberg ldquoHigh incidence of plantgrowth-stimulating bacteria associated with the rhizosphere ofwheat grown on salinated soil in Uzbekistanrdquo EnvironmentalMicrobiology vol 10 no 1 pp 1ndash9 2008

[4] R Hayat S Ali U Amara R Khalid and I Ahmed ldquoSoilbeneficial bacteria and their role in plant growth promotion areviewrdquoAnnals ofMicrobiology vol 60 no 4 pp 579ndash598 2010

[5] R Marasco E Rolli B Ettoumi et al ldquoA drought resistance-promoting microbiome is selected by root system under desertfarmingrdquo PLoS ONE vol 7 no 10 Article ID e48479 2012

[6] S K Upadhyay D P Singh and R Saikia ldquoGenetic diver-sity of plant growth promoting rhizobacteria isolated fromrhizospheric soil of wheat under saline conditionrdquo CurrentMicrobiology vol 59 no 5 pp 489ndash496 2009

[7] S Mayak T Tirosh and B R Glick ldquoPlant growth-promotingbacteria confer resistance in tomato plants to salt stressrdquo PlantPhysiology and Biochemistry vol 42 no 6 pp 565ndash572 2004

[8] K C Ravindran K Venkatesana V Balakrishnana K P Chel-lappana and T Balasubramanian ldquoRestoration of saline land byhalophytes for Indian soilsrdquo Soil Biology and Biochemistry vol39 pp 2661ndash2664 2007

[9] D M Al-Mailem N A Sorkhoh M Marafie H Al-AwadhiM Eliyas and S S Radwan ldquoOil phytoremediation potentialof hypersaline coasts of the Arabian Gulf using rhizospheretechnologyrdquo Bioresource Technology vol 101 no 15 pp 5786ndash5792 2010

[10] E Manousaki and N Kalogerakis ldquoHalophytes present newopportunities in phytoremediation of heavy metals and salinesoilsrdquo Industrial and Engineering Chemistry Research vol 50no 2 pp 656ndash660 2011

[11] E P Glenn J W OrsquoLeary M C Watson T L Thompson andR O Kuehl ldquoSalicornia bigelovii Torr an oilseed halophyte forseawater irrigationrdquo Science vol 251 no 4997 pp 1065ndash10671991

[12] F M Attia A A Alsobayel M S Kriadees M Y Al-Saiadyand M S Bayoumi ldquoNutrient composition and feeding valueof Salicornia bigelovii torr meal in broiler dietsrdquo Animal FeedScience and Technology vol 65 no 1ndash4 pp 257ndash263 1997

[13] United Nations Secretary-Generalrsquos High-level Panel on GlobalSustainability Resilient People Resilient Planet A Future WorthChoosing United Nations New York NY USA 2012

[14] C J Ruan J A T da Silva S Mopper Q Pei and S LuttsldquoHalophyte improvement for a salinizedworldrdquoCritical Reviewsin Plant Sciences vol 29 no 6 pp 329ndash359 2010

[15] D Ramadoss V K Lakkineni P Bose S Ali and K Anna-purna ldquoMitigation of salt stress in wheat seedlings by halotol-erant bacteria isolated from saline habitatsrdquo SpringerPlus vol 2no 1 article 6 2013

[16] S Tiwari P Singh R Tiwari et al ldquoSalt-tolerant rhizobacteria-mediated induced tolerance in wheat (Triticum aestivum) andchemical diversity in rhizosphere enhance plant growthrdquo Biol-ogy and Fertility of Soils vol 47 no 8 pp 907ndash916 2011

[17] M Argandona R Fernandez-Carazo I Llamas et al ldquoThemoderately halophilic bacterium Halomonas maura is a free-living diazotrophrdquo FEMS Microbiology Letters vol 244 no 1pp 69ndash74 2005

[18] Y Bashan MMoreno and E Troyo ldquoGrowth promotion of theseawater-irrigated oilseed halophyte Salicornia bigelovii inoc-ulated with mangrove rhizosphere bacteria and halotolerantAzospirillum spprdquo Biology and Fertility of Soils vol 32 no 4pp 265ndash272 2000

[19] I Gontia K Kavita M Schmid A Hartmann and B JhaldquoBrachybacterium saurashtrense sp nov a halotolerant root-associated bacterium with plant growth-promoting potentialrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 61 part 12 pp 2799ndash2804 2011

[20] T Ozawa J Wu and S Fujii ldquoEffect of inoculation witha strain of Pseudomonas pseudoalcaligenes isolated from theendorhizosphere of Salicornia europea on salt tolerance of theglasswortrdquo Soil Science and Plant Nutrition vol 53 no 1 pp12ndash16 2007

[21] E Rueda-Puente T Castellanos E Troyo-Dieguez J L D deLeon-Alvarez and B Murillo-Amador ldquoEffects of a nitrogen-fixing indigenous bacterium (Klebsiella pneumoniae) on thegrowth and development of the halophyte Salicornia bigelovii asa new crop for saline environmentsrdquo Journal of Agronomy andCrop Science vol 189 no 5 pp 323ndash332 2003

[22] B Jha I Gontia and A Hartmann ldquoThe roots of the halo-phyte Salicornia brachiata are a source of new halotolerantdiazotrophic bacteria with plant growth-promoting potentialrdquoPlant and Soil vol 356 no 1-2 pp 265ndash277 2012

[23] Forssk Fl Aeg Arab 2 1775[24] E Le Flocrsquoh L Boulos and E Vela Catalogue synonymique

commente de la flore de Tunisie Republique tunisienne min-istere de lrsquoenvironnement et du developpement durable banquenationale de genes 2010

[25] C Schbereiter-Gurtner G Pinar W Lubitz and S Rolleke ldquoAnadvanced molecular strategy to identify bacterial communitieson art objectsrdquo Journal of Microbiological Methods vol 45 no2 pp 77ndash87 2001

[26] G Muyzer E C de Waal and A G Uitterlinden ldquoProfilingof complex microbial populations by denaturing gradient gel

12 BioMed Research International

electrophoresis analysis of polymerase chain reaction-amplifiedgenes coding for 16S rRNArdquo Applied and Environmental Micro-biology vol 59 no 3 pp 695ndash700 1993

[27] M Cardinale L Brusetti P Quatrini et al ldquoComparison ofdifferent primer sets for use in automated ribosomal intergenicspacer analysis of complex bacterial communitiesrdquo Applied andEnvironmentalMicrobiology vol 70 no 10 pp 6147ndash6156 2004

[28] D Daffonchio S Borin G Frova P L Manachini and CSorlini ldquoPCR fingerprinting of whole genomes the spacersbetween the 16S and 23S rRNA genes and of intergenic tRNAgene regions reveal a different intraspecific genomic variabilityof Bacillus cereus and Bacillus licheniformisrdquo International Jour-nal of Systematic Bacteriology vol 48 pp 107ndash116 1998

[29] J M Bric R M Bostock and S E Silverstone ldquoRapid in situassay for indoleacetic acid production by bacteria immobilizedon a nitrocellulose membranerdquo Applied and EnvironmentalMicrobiology vol 57 no 2 pp 535ndash538 1991

[30] F Ahmad I Ahmad and M S Khan ldquoScreening of free-livingrhizospheric bacteria for their multiple plant growth promotingactivitiesrdquo Microbiological Research vol 163 no 2 pp 173ndash1812008

[31] J C Cappuccino and N Sherman ldquoNegative stainingrdquo inMicrobiology A Laboratory Manual J C Cappuccino and NSherman Eds pp 125ndash179 BenjaminCummings RedwoodCity Calif USA 3rd edition 1992

[32] P Nielsen and J Soslashrensen ldquoMulti-target and medium-independent fungal antagonism by hydrolytic enzymes inPaenibacillus polymyxa and Bacillus pumilus strains from barleyrhizosphererdquo FEMSMicrobiology Ecology vol 22 no 3 pp 183ndash192 1997

[33] D M Penrose and B R Glick ldquoMethods for isolating and char-acterizingACCdeaminase-containing plant growth-promotingrhizobacteriardquo Physiologia Plantarum vol 118 no 1 pp 10ndash152003

[34] F Poly L J Monrozier and R Bally ldquoImprovement in the RFLPprocedure for studying the diversity of nifH genes in communi-ties of nitrogen fixers in soilrdquo Research in Microbiology vol 152no 1 pp 95ndash103 2001

[35] L Lambertsen C Sternberg and S Molin ldquoMini-Tn7 trans-posons for site-specific tagging of bacteria with fluorescentproteinsrdquo EnvironmentalMicrobiology vol 6 no 7 pp 726ndash7322004

[36] WT Liu T LMarshHCheng and L J Forney ldquoCharacteriza-tion of microbial diversity by determining terminal restrictionfragment length polymorphisms of genes encoding 16S rRNArdquoApplied and Environmental Microbiology vol 63 no 11 pp4516ndash4522 1997

[37] M M Fisher and E W Triplett ldquoAutomated approach forribosomal intergenic spacer analysis of microbial diversity andits application to freshwater bacterial communitiesrdquo Appliedand Environmental Microbiology vol 65 no 10 pp 4630ndash46361999

[38] C A Lozupone and R Knight ldquoGlobal patterns in bacterialdiversityrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 104 no 27 pp 11436ndash11440 2007

[39] K Smalla GWieland A Buchner et al ldquoBulk and rhizospheresoil bacterial communities studied by denaturing gradientgel electrophoresis plant-dependent enrichment and seasonalshifts revealedrdquo Applied and Environmental Microbiology vol67 no 10 pp 4742ndash4751 2001

[40] T M Caton L R Witte H D Ngyuen J A Buchheim MA Buchheim and M A Schneegurt ldquoHalotolerant aerobic

heterotrophic bacteria from the Great Salt Plains of OklahomardquoMicrobial Ecology vol 48 no 4 pp 449ndash462 2004

[41] J Tang A P Zheng E S P Bromfield et al ldquo16S rRNAgene sequence analysis of halophilic and halotolerant bacteriaisolated from a hypersaline pond in Sichuan Chinardquo Annals ofMicrobiology vol 61 no 2 pp 375ndash381 2011

[42] R R de la Haba D R Arahal M C Marquez and A VentosaldquoPhylogenetic relationships within the family Halomonadaceaebased on comparative 23S and 16S rRNA gene sequenceanalysisrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 60 no 4 pp 737ndash748 2010

[43] A C F Dias F D Andreote F Dini-Andreote et al ldquoDiversityand biotechnological potential of culturable bacteria fromBrazilian mangrove sedimentrdquo World Journal of Microbiologyand Biotechnology vol 25 no 7 pp 1305ndash1311 2009

[44] K A El-Tarabily and T Youssef ldquoEnhancement of morpholog-ical anatomical and physiological characteristics of seedlingsof the mangrove Avicennia marina inoculated with a nativephosphate-solubilizing isolate of Oceanobacillus picturae undergreenhouse conditionsrdquo Plant and Soil vol 332 no 1 pp 147ndash162 2010

[45] B E Michel and M R Kaufmann ldquoThe osmotic potential ofpolyethylene glycol 6000rdquo Plant Physiology vol 51 no 5 pp914ndash917 1973

[46] N P Hua A Hamza-Chaffai R H Vreeland H Isoda andT Naganuma ldquoVirgibacillus salarius sp nov a halophilicbacterium isolated from a Saharan salt lakerdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 58 no10 pp 2409ndash2414 2008

[47] T Quesada M Aguilera J A Morillo A Ramos-Cormenzanaand M Monteoliva-Sanchez ldquoVirgibacillus olivae sp novisolated from waste wash-water from processing of Spanish-style green olivesrdquo International Journal of Systematic andEvolutionary Microbiology vol 57 no 5 pp 906ndash910 2007

[48] I Romano L Lama P Orlando B Nicolaus A Giordanoand A Gambacorta ldquoHalomonas sinaiensis sp nov a novelhalophilic bacterium isolated from a salt lake inside RasMuhammad Park Egyptrdquo Extremophiles vol 11 no 6 pp 789ndash796 2007

[49] C Sanchez-Porro R R de la Haba N Soto-Ramırez M CMarquez R Montalvo-Rodrıguez and A Ventosa ldquoDescrip-tion ofKushneria aurantia gen nov sp nov a novelmember ofthe family Halomonadaceae and a proposal for reclassificationof Halomonas marisflavi as Kushneria marisflavi comb nov ofHalomonas indalinina as Kushneria indalinina comb nov andof Halomonas avicenniae as Kushneria avicenniae comb novrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 59 no 2 pp 397ndash405 2009

[50] N Soto-Ramırez C Sanchez-Porro S Rosas et al ldquoHalomonasavicenniae sp nov isolated from the salty leaves of the blackmangrove Avicennia germinans in Puerto Ricordquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no5 pp 900ndash905 2007

[51] S Compant B Duffy J Nowak C Clement and E A BarkaldquoUse of plant growth-promoting bacteria for biocontrol ofplant diseases principles mechanisms of action and futureprospectsrdquoApplied and Environmental Microbiology vol 71 no9 pp 4951ndash4959 2005

[52] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects for

BioMed Research International 13

utilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[53] R S Redman Y O Kim C J D A Woodward et alldquoIncreased fitness of rice plants to abiotic stress via habitatadapted symbiosis a strategy for mitigating impacts of climatechangerdquo PLoS ONE vol 6 no 7 Article ID e14823 2011

[54] S Timmusk V Paalme T Pavlicek et al ldquoBacterial distributionin the rhizosphere of wild barley under contrasting microcli-matesrdquo PLoS ONE vol 6 no 3 Article ID e17968 2011

[55] M A Siddikee P S Chauhan R Anandham G H Han andT Sa ldquoIsolation characterization and use for plant growthpromotion under salt stress of ACC deaminase-producinghalotolerant bacteria derived from coastal soilrdquo Journal ofMicrobiology and Biotechnology vol 20 no 11 pp 1577ndash15842010

[56] V Sgroy F Cassan O Masciarelli M F del Papa A LagaresandV Luna ldquoIsolation and characterization of endophytic plantgrowth-promoting (PGPB) or stress homeostasis-regulating(PSHB) bacteria associated to the halophyte Prosopis strombu-liferardquoAppliedMicrobiology and Biotechnology vol 85 no 2 pp371ndash381 2009

[57] M Bacilio H Rodriguez M Moreno J P Hernandez and YBashan ldquoMitigation of salt stress in wheat seedlings by a gfp-taggedAzospirillum lipoferumrdquoBiology and Fertility of Soils vol40 no 3 pp 188ndash193 2004

[58] B Fan X H Chen A Budiharjo W Bleiss J Vater andR Borriss ldquoEfficient colonization of plant roots by the plantgrowth promoting bacteriumBacillus amyloliquefaciens FZB42engineered to express green fluorescent proteinrdquo Journal ofBiotechnology vol 151 no 4 pp 303ndash311 2011

[59] B Fan R Borriss W Bleiss and X Wu ldquoGram-positive rhi-zobacterium Bacillus amyloliquefaciens FZB42 colonizes threetypes of plants in different patternsrdquo Journal ofMicrobiology vol50 no 1 pp 38ndash44 2012