Hindawi Publishing Corporation e Scientic World Journal Volume , Article ID  ,  pages http://dx.doi.org/.// 

Research Article Genotypic Characterization of Azotobacteria Isolated from Argentinean Soils and Plant-Growth-Promoting Traits of Selected Strains with Prospects for Biofertilizer Production

Esteban Julián Rubio,1 Marcela Susana Montecchia,2 Micaela Tosi,2 Fabricio Darío Cassán,3 Alejandro Perticari,1 and Olga Susana Correa2

 Instituto de Microbiolog´ıa y Zoolog´ıa Agr´ıcola (IMYZA), Instituto Nacional de Tecnolog´ıa Agropecuaria (INTA), Dr. Nicolas´ Repetto y De los Reseros s/n,  Hurlingham, Buenos Aires, Argentina  Catedra´ de Microbiolog´ıa Agr´ıcola/INBA (CONICET/UBA), Facultad de Agronom´ıa, Universidad de Buenos Aires, Av San Mart´ın , C DSE Ciudad Autonoma´ de Buenos Aires, Argentina  Laboratorio de Fisiolog´ıa Vegetal, Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, F´ısico-Qu´ımicas y Naturales, Universidad Nacional de R´ıo Cuarto, Ruta , Km  ,  R´ıo Cuarto, Cordoba,´ Argentina

Correspondence should be addressed to Olga Susana Correa; [email protected]

Received  August ; Accepted September 

Academic Editors: C. Bystro and H. Freitas

Copyright ©  Esteban Julian´ Rubio et al. is 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.

e genetic diversity among  putative isolates obtained from agricultural and non-agricultural soils was assessed using rep-PCR genomic ngerprinting and identied to species level by ARDRA and partial  S rRNA gene sequence analysis. High diversity was found among the isolates, identied as A. chroococcum, A. salinestris, and A. armeniacus. Selected isolates were char- acterized on the basis of phytohormone biosynthesis, nitrogenase activity, siderophore production, and phosphate solubilization.

Indole- acetic-acid (IAA), gibberellin (GA3) and zeatin (Z) biosynthesis, nitrogenase activity, and siderophore production were found in all evaluated strains, with variation among them, but no phosphate solubilization was detected. Phytohormones excreted −1 −1 −1 to the culture medium ranged in the following concentrations: .– . g IAA mL , .–. g GA3 mL , and .–. g Z mL . Seed inoculations with further selected Azotobacter strains and treatments with their cell-free cultures increased the number of seminal roots and root hairs in wheat seedlings. is latter e ect was mimicked by treatments with IAA-pure solutions, but it was not related to bacterial root colonization. Our survey constitutes a rst approach to the knowledge of Azotobacter species inhabiting Argentinean soils in three contrasting geographical regions. Moreover, this phenotypic characterization constitutes an important contribution to the selection of Azotobacter strains for biofertilizer formulations.

1. Introduction positive e ects on plant growth, mechanisms involved are not

fully understood. e ability to x N2 was the main feature e genus Azotobacter, which belongs to the family Pseu- leading to the use of Azotobacter as a biofertilizer in the past. domonadaceae from the subclass -, com- Nowadays, however, it is well established that non-symbiotic prises seven species: Azotobacter vinelandii, A. chroococcum, xation can improve plant growth only indirectly, by increas- A. salinestris, A. nigricans, A. beijerinckii, A. paspali, and A. ing soil nitrogen aer mineralization of N-xers’ biomass. armeniacus []. Azotobacteria are aerobic, heterotrophic, and More likely, additional abilities of azotobacteria, such as free-living N2-xing , which can be isolated from soil, phosphate solubilization and phytohormone and siderophore water, and sediments []. Several studies have demonstrated synthesis, might contribute more directly to increase plant that seed inoculation with Azotobacter improves maize [], growth and crop yield [, , ]. wheat [, ], and rice [ ] yields. However, although there Like many plant-growth promoting bacteria, azotobac- is a considerable amount of experimental evidence of these teria have the capacity to excrete auxins to the culture  e Scientic World Journal medium. Auxins and indole- acetic-acid (IAA) as the Isolates were preserved at − ∘C in Burk’s medium [ ] with most common member of auxin family were the rst plant % (v/v) glycerol. hormones to be discovered and are implicated in virtually Azotobacter vinelandii reference strains (NRRL B-  , every aspect of plant growth and development. It has been NRRL B- , and NRRL B- ) were obtained from the reported that inoculation with auxin-releasing Azotobacter ARS Culture Collection (NRRL), USA, and A. chroococcum strains increases growth, yield, and nitrogen uptake in wheat reference strain BNM  , isolated from Argentinian soils, and maize and that the combined application of Azotobacter was provided by the Banco Nacional de Microorganismos, and tryptophan, which is oen implicated in IAA synthesis, Argentina. enhances plant growth in a greater extent [, , ]. ese Electrical conductivity (EC), organic matter (OM), pH, results suggest that auxin production might be a key mech- and extractable phosphorus of the soils samples were deter- anism of Azotobacter in promoting plant growth and yield, as mined at the Instituto de Suelos (INTA, Buenos Aires, it has been reported in other bacteria. Argentina) using standard procedures []. e importance of studying plant-growth promoting bac- teria (PGPR) lies on their potential to be used as biofertilizers. .. Rep-PCR Genomic Fingerprinting. Repetitive sequence- e use of biofertilizers containing living microorganisms is based PCR genomic ngerprints of isolates were obtained a welcoming management alternative in sustainable systems, with BOX-AR primers [] as previously described [], by like organic and low-input agriculture, as well as a tool to using -L portions of whole-cell suspensions of each isolate reduce the use of chemicals in intensive agriculture []. as templates. Fingerprints were analyzed using GelCompar When formulating a biofertilizer, it is highly recommended II v. . (Applied Maths NV). Dendrogram was elaborated to consider the use of native bacteria, because they are better based on Pearson’s correlation coecient and the UPGMA adapted to ecological conditions and, therefore, are more algorithm. competitive than nonnative strains []. Hence, the isolation and characterization of native bacterial strains should be one of the rst steps when developing commercial biofertilizers. .. Ampli ed Ribosomal DNA Restriction Analysis (ARDRA). In Argentina, the diversity of Azotobacter in soils has not yet Representative strains of each rep-PCR cluster were analyzed been studied and any Azotobacter-based biofertilizers have by ARDRA, as previously described [], using the primers fD been developed. and rD and the restriction enzymes RsaI orHha I. ARDRA For the above mentioned facts, the aims of our study proles were analyzed with GelCompar II and compared were to isolate and characterize Azotobacter strains from usingtheDicesimilaritycoecienttoconstructthesimilarity agricultural and non-agricultural soils, covering a wide range matrix. e dendrogram was obtained by UPGMA. of geographic regions and soil types, and to study some In silico ARDRA was carried out with HhaI using the bacterial traits involved in plant growth stimulation. To restriction mapper soware (http://www.restrictionmapper test this, we rst assessed genetic diversity among isolates .org/) and  S rRNA gene sequences AB   (A. salinestris T by repetitive sequence-based PCR genomic ngerprinting ATCC  4 ) and FJ (A. salinestris I-A), both ob- (rep-PCR) and identied them by amplied ribosomal DNA tained from GenBank. restriction analysis (ARDRA) and partial  S rRNA gene sequence analysis. en, some of these isolated strains were .. S rRNA Gene Sequencing. e partial  S rRNA gene tested for hormone biosynthesis (indole--acetic acid (IAA), sequence was amplied using primers Y and Y []. en, gibberellic acid (GA ), and zeatin (Z)), siderophore produc- 3 amplicons (∼  bp) were puried using the QIAquick PCR tion, nitrogen xation capacity, and phosphate solubilization. purication kit (Qiagen, GmbH) and sequenced by Unidad Finally, we tested early-growth stimulation of wheat roots by de Genomica´ (Instituto de Biotecnolog´ıa, INTA, Buenos inoculation with some of the isolated Azotobacter strains. Aires, Argentina) in both directions using the same primers. e obtained sequences were compared with those from 2. Materials and Methods GenBank using BLASTN .. [ ]. .. Soil Sampling, Bacterial Isolation, and Azotobacter Refer- ence Strains. In total,  bulk soil samples (– cm) were .. Nucleotide Sequence Accession Numbers. e obtained collected from agricultural ( samples) and non-agricultural  S rRNA gene sequences were deposited at the Gen- sites ( samples) during spring  . Samples belonged to Bank/EMBL/DDBJ database under the following acces-  di erent locations of Northwest, Pampas, and Patagonia sion numbers: HQ , HQ  , HQ  , HQ  , regions of Argentina (see Supplementary Material available HQ  , HQ  , and HQ  . online at http://dx.doi.org/.// ). Soil aggre- gates (∼ mm) were spread onto the surface of Petri dishes .. Determination of Potential Plant Growth-Promoting containing N-free Burk’s agar medium with mannitol as Traits. Eighteen selected strains were assessed for sidero- C-source []. Aer ve days at  ∘C, slimy and glistening phore production according to the O-CAS method [ ]. Azotobacter-like colonies growing around soil particles were Phosphate-solubilizing activity was tested on Pikovskaya selected and further puried in N-free LG with bromothymol medium [ ], NBRIP medium [ ] and modied Burk’s agar blue agar medium []. Motility, pigment production, and medium [], adding .% of Ca3(PO4)2 to each medium as encystment were determined as previously described []. insoluble P source. In both assays, Pseudomonas uorescens e Scientic World Journal 

BNM (Banco Nacional de Microorganismos, Buenos e e ects on root tip morphology of cell-free culture Aires, Argentina) was used as a positive control. of two selected A. salinestris strains (AT and AT ) with Auxin production was determined using a colorimetric di erent levels of phytohormone production (Figure ) and assay [], with measurements aer , , , and  days of root colonization (Table ) but similar nitrogenase activity growth in modied LG (LGSP) liquid medium containing % (Figure ) were assessed and compared to the application and .% soymeal peptone. At each time interval, the of two IAA-pure solutions,  and  gmL−1. Fieen pre- number of cells (cfu mL−1) was determined by plate counting germinated wheat seeds per treatment were placed in three on LG agar. Petri dishes (ve seeds per dish) containing . % water agar. Nitrogenase activity was estimated by the acetylene Seedling treatments were as follows: (a) control ( L of reduction assay. Bacterial cultures were grown in N-free sterile distilled water), (b)  LofgmL−1 IAA-pure ∘ Burk’s agar medium at  C for  h and ethylene production solution, (c)  LofgmL−1 IAA-pure solution, (d) was measured by gas chromatography [], using a Hewlett  L ofA. salinestris AT cell-free culture, and (e)  L of Packard Series II   equipped with a ame ionization A. salinestris AT cell-free culture. Aer  days at ∘C under detector (FID) and a stainless-steel Porapak N column dark conditions, seedling roots were stained with crystal (. mm ×  m; / mesh). e injector, oven, and detec- violet solution (. % in % ethanol) and observed in a tor temperatures were ∘C, ∘C, and ∘C, respectively. binocular microscope at x. −1 N2 was used as carrier gas (. cm s linear gas velocity). Total protein concentration of bacterial cells was determined .. Experimental Design and Data Analysis. Each inocula- by the Lowry method with the DC Protein Assay kit (Bio- tion experiments were performed in a complete randomized Rad, USA). Nitrogenase activity was expressed as mmol design. Data were analyzed by ANOVA and DGC multiple ethylene produced per mg of protein in  h. comparisons post hoc analysis []( = 0.05), using Indole--acetic acid (IAA), gibberellic acid (GA3), and INFOSTAT soware []. zeatin (Z) production were determined for six selected Azoto- bacter spp. strains grown in LGSP liquid medium at  ∘C for days. Z was identied and quantied by HPLC-UV, whereas 3. Results IAA and GA were identied by gas chromatography-mass 3 .. Azotobacter Isolates Obtained from Argentinean Soils and spectrometry with selective ion monitoring (GC-MS-SIM), Chemical Parameters of Soils. We isolated Azotobacter-like as previously described []. bacteria from  soil samples ( agricultural and  non- agricultural soils) from a total of  screened samples (Table  . . E ects of Azotobacter Inoculation and IAA Pure Solutions and Supplementary Material). Isolates were obtained from on the Number of Seminal Roots and Root Hairs of Wheat soils with a wide range of values for organic matter con- Seedlings. For plant tests, seeds of wheat (Triticum aestivum tent (. –. %), pH (. – . ), electrical conductivity (.– cv. Baguette Premium , Nidera, Buenos Aires, Argentina) . mS cm−1), and extractable phosphorus (. – . ppm) were surface-disinfected (% NaClO for  minutes) and ger- (Table ). minated in plastic containers ( ×  ×  cm) on lter paper We obtained  bacterial isolates that were preliminary soaked with sterile distilled water. To maintain humidity, characterized on the basis of pigment production and cell containers were wrapped in transparent plastic bags and morphology. All of them produced nondi usible brown placed in a growth chamber at ∘C with a  h light/ h dark pigments in agar medium, showed motile cells, formed cysts regime for  h. For inoculation, bacterial strains were grown in butanol-containing medium, and showed no uorescent in LGSP liquid medium at  ∘C for days (∼ 8 cfu mL−1). pigments under UV light (data not shown). Fieen pregerminated seeds were inoculated with  L of bacterial culture (∼7 cells) per seed and grown for .. Genomic Fingerprinting by rep-PCR. e intraspecic days as described above. Eight treatments were applied: (a) diversity among  isolates was assessed by means of rep-PCR. control ( L of sterile distilled water); (b) and (c) two phy- Most isolates showed distinctive banding proles, reecting tohormone treatments based on  L of low ( gmL−1) the genetic diversity among them. e cluster analysis of and high ( gmL−1) concentrations of pure-IAA solutions ngerprints revealed six major groups among all isolates at (Sigma-Aldrich), sterilized by ltration (. m lter); (d) % similarity level (Figure ). Isolates showing highly similar A. salinestris AT ; (e) A. salinestris AT ; (f) A. salinestris ngerprints (similarity > %) were considered clonemates. AT ; (g) A. chroococcum AT; and (h) A. chroococcum As a result,  distinct strains were obtained. No clear rela- AT. Treatments were run in triplicate (three containers tionship could be established between rep-PCR clustering each). For bacterial root colonization, roots of two plants per and the geographical origin of isolates. For example, group container (a total of six plants per treatment) were ground in  included strains which were isolated from four provinces  mL of sterile distilled water with mortar and pestle. Serial (Buenos Aires, Chubut, Entre R´ıos, and Jujuy) of the three dilutions were inoculated in triplicate on LG agar plates and regions (Pampas, Northwest, and Patagonia). However, some incubated at  ∘C for  h. At the end of the experiment, root tendencies between clustering and the origin of soil samples colonization (cfu per root of Azotobacter-like colonies) and were observed. Group  clustered all isolates from Cordoba´ number of seminal roots were determined. Two independent province (Pampas region), group  included strains isolated experiments were run. from Salta and Santiago del Estero provinces (Northwest  e Scientic World Journal

Similarity (%) 20 40 60 80 100

AT4 AT27 AT26 BNM 272 A.chroococcumm AT13 AT28 AT25 AT39 AT30 AT43 1 AT31 AT11 AT24 AT5 AT9 AT22 AT32 2 AT33 AT36 AT1 AT2 AT16 AT17 3 AT18 AT14 AT19 4 AT29 AT42 AT37 5 AT38 AT12 6 AT10

F : Genetic diversity of azotobacteria isolated from agricultural and non-agricultural soils from di erent regions of Argentina revealed by rep-PCR genomic ngerprinting analysis. e dendrogram was constructed by using the Pearson correlation coecient () and the UPGMA method using GelCompar II version . soware. e groups indicated by  to numbers were dened at the % similarity level (vertical dashed line). e cophenetic correlation value for this dendrogram was . .

region), and group  included two strains obtained from cluster I showed proles distinctive of A. chroococcum, as Chubut province (Patagonia region) (Figure  and Table ). reported by Aquilanti et al.  [], and identical to those of We chose representative strains of each group to classify them A. chroococcum reference strain BNM  , they were assigned using ARDRA. to this species. Cluster II included only strain AT, which showed a characteristic banding prole of the species A. .. ARDRA and S rRNA Gene Sequence Analysis. ARDRA armeniacus [], whereas cluster III contained only the three with RsaI andHha I restriction enzymes was used to identify A. vinelandii strains used as reference. e ARDRA proles of strains in cluster IV, obtained experimentally, were similar Azotobacter strains to genus and species level, as previ- T ously recommended for the molecular identication of these to those of A. salinestris reference strains ATCC   and microorganisms []. e  chosen strains represented, I-A done in silico. According to these results, the strains altogether, the six rep-PCR clusters. All strains yielded single of heterogeneous cluster IV (Figure ) were assigned to A. amplication products of the expected size (about , bp) salinestris. for the  S rRNA genes and showed identical restriction To conrm species identication of isolates, partial RsaI proles (data not shown), characteristic of the genus sequencing of the  S rRNA gene was performed for seven Azotobacter [, ]. When ARDRA was performed using strains representing ARDRA clusters. Based on the similarity HhaI, six di erent proles were obtained. Cluster analysis observed among these sequences, strains AT and AT in of HhaI restriction proles revealed four distinct clusters at cluster I (Figure ) were related to A. chroococcum LMG T % similarity level (Figure ). Since all strains grouped in  ( % identity), strain AT in cluster II was related e Scientic World Journal  ∗∗ ) P (ppm)  − es and soil chemical Soil chemical parameters . . . .  . . . . . . . . . . .  .  . . nd  .  . .  . . . . . .  . .  . . . . . .  . . . .  .  .  . . .  .  .  . .  .  . . .  . . . . . . . . . .  .  . . .  . . . . . OM (%) pH EC (mS cm A. salinestris A. salinestris A. salinestris A. armeniacus A. armeniacus A. armeniacus A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. chroococcum Species assignments sequence S rDNA (accession number) Partial isolates were obtained. Summary of ngerprinting and identication results of isolat I I HQ   I I HQ   I I I I II HQ   IV HQ  IV nd nd nd nd nd nd nd nd nd nd Azotobacter ARDRA cluster m which AT  AT  AT  AT  a River bank AT  Urban land AT  Wheat crop AT  Side of road AT  Wheat crop  AT  Sampling site Isolate rep-PCR group Wheat crop  AT  Wheat crop  AT  Side of route Soybean crop AT  Maize stubble AT  Lagoonbank AT  Lagoon bank  AT  Soybean crop  AT  Natural pasture AT  Natural pasture AT  Natural pasture AT  Natural pasture AT  Agriculturalbare AT  ı) ´ ın V. ´ ´ on) ıos ´ ın) ´ ´ a) ∗ ´ alez) ´ ´ ´ ordoba ordoba ordoba Chubut (Esquel) Chubut (Gaiman) Buenos Aires (Mar Chiquita) Buenos Aires (Santa Clara del Mar) Buenos Aires (Mar Chiquita) Buenos Aires (Balcarce) Geographical origin Buenos Aires (Azul) Buenos Aires (Santa Clara del Mar) Chubut (Trevel Jujuy (Tilcara) Salta (Embarcaci Gonz Salta (Joaqu Santiago del Estero (Quimil Jujuy (Tilcara) (Paran Entre R C (Corral de Bustos) C (Corral de Bustos) C (Corral de Bustos) T : Geographical origin and land use of soil samples fro characteristics. e Scientic World Journal ∗∗ ) P (ppm)  − Soil chemical parameters .  .  . . . . . . . . . .  . . . .  . . . . . . .  . . . nd . OM (%) pH EC (mS cm t region, Chubut is a province from Patagonia region of A. salinestris A. salinestris A. salinestris A. salinestris A. salinestris A. salinestris A. salinestris A. salinestris A. salinestris A. salinestris Species assignments sequence S rDNA (accession number) Partial lta, and Santiago del Estero are provinces from the Northwes T : Continued. IV HQ  IV IV HQ  IV IV HQ IV IV nd nd nd ARDRA cluster ble phosphorus; nd: not determined. AT  AT  AT  AT AT  a a ıos, and Santa Fe are provinces of the Pampas region; Jujuy, Sa ´ River bank AT  Sampling site Isolate rep-PCR group Side of route Side of route Soybean crop  AT  Soybean crop  AT  Soybean crop  AT  Natural pasture AT  ´ ordoba, Entre R ∗ OM: organic matter; EC: electrical conductivity; P: extracta Buenos Aires, C Corresponded to the same soil sample. Geographical origin Santa Fe (Videla) Santa Fe (Videla) Chubut (Puerto Madryn) Chubut (Villa Ameghino) Jujuy (Tilcara) Jujuy (Tilcara) Argentina. a ∗∗ ∗ e Scientic World Journal

Similarity (%) 60 80 100

AT4 AT9 AT25 AT27 AT28 AT30 AT43 I AT31 BNM 272 A.chroococcumm II AT33 NRRL B-14627A.vinelandii III NRRL B-14641A.vinelandii NRRL B-14644 A.vinelandii AT12 AT38 AT37 AT10 AT14 AT29 AT19 IV AT42 AT18

F : Amplied ribosomal DNA restriction analysis (ARDRA) of Azotobacter representative strains of each rep-PCR group and reference strains. e dendrogram based on analysis of restriction patterns of  S rDNA obtained with HhaI was built using the GelCompar II program and the Dice () pairwise coecient of similarity and the UPGMA algorithm. Clusters were dened at the  > 80% similarity level. e cophenetic correlation value for this dendrogram was . .

T to A. armeniacus DSM   ( % identity), and the four observed aer two and three days of growth, without any strains in cluster IV (AT , AT , AT , and AT) were −1 T changes in cfu mL (data not shown). Finally, bacterial related to A. salinestris ATCC   ( -% identity). strains di ered in the levels of auxin excreted to the culture Summarizing, according to the results obtained by rep- medium at the end of the assay, covering a range of values PCR, ARDRA, and partial sequencing of the  S ribosomal from . to  . gmL−1 (Table ). A. salinestris AT, AT, gene, the  isolates of group  of rep-PCR (Figure ) were AT , and AT and A. chroococcum AT, AT, AT, and classied as A. chroococcum, the three isolates of group  as AT reached up to a ∼-fold increase from the rst to the A. armeniacus, and the  isolates included in groups  to as h day (Table ). No changes in the number (cfu mL−1) A. salinestris. of bacteria were observed at the end of the assay (data not shown). .. Siderophore and Phytohormone Production, Phosphate Using these results, the  Azotobacter strains were Solubilization, and Nitrogenase Activity. All the  strains arbitrarily classied as low- (– gmL−1), medium- ( – tested exhibited a color change from blue to orange in  gmL−1), and high- (> gmL−1) auxin producers CAS medium, which is indicative of siderophore production. (Table ). en, we selected three low-auxin-producing Phosphate-solubilizing activity was not evident in any of the strains (AT , AT , and AT) and three high-auxin- Azotobacter strains assayed, independently of the medium producing strains (AT , AT, and AT) and assessed used (data not shown). All preselected strains were assayed them in nitrogen xing capacity and biosynthesis of three for auxin production in LGSP medium using the Salkowski phytohormones (IAA, GA3, and Z). reagent method. Aer one day of growth (∼8 cfu mL−1), all Concerning the nitrogenase activity, the highest activity bacterial strains produced low levels of auxin (. gmL−1 −1 −1  levels (∼ mmol C2H4 mg protein  h ) were exhibited to .  gmL−1) (Table ). An important increase was by A. salinestris AT and A. chroccoccum AT strains. e Scientic World Journal

T : Auxin production capacity of representative Azotobacter strains as determined by a colorimetric assay using the Salkowski reagent and results of rep-PCR clustering.

rep-PCR Auxinproduction( g mL− ) Strain  group Day  Day  Day  Day  a a c c A. chroococcum AT  1.80 ± 0.64 2.50 ± 0.27 4.13 ± . 4.23 ± . a a a b A. chroococcum AT  1.10 ± 0.41 4.97 ± 1.27 12.26 ± . 12.34 ± . a a a b A. chroococcum AT  1.41 ± 0.83 4.58 ± 1.37 11.38 ± . 12.72 ± . a a a b A. chroococcum AT  2.08 ± 0.09 8.54 ± 2.02 12.62 ± .  13.25 ± . a a a b A. chroococcum AT  1.38 ± 0.61 9.30 ± 0.60 12.68 ± .  14.76 ± . a a a b A. chroococcum AT  2.16 ± 0.81 7.81 ± 0.44 14.80 ± . 14.81 ± .  a a a b A. chroococcum AT  2.64 ± 0.57 7.97 ± 2.31 12.82 ± 0.07 15.33 ± . a a a b A. chroococcum AT  1.59 ± 0.40 6.01 ± 1.09 12.92 ± 1.40 15.36 ± . a a a b A. chroococcum AT  1.40 ± 0.60 4.97 ± 1.27 12.86 ± 0.60 16.11 ± . a a b b A. armeniacus AT  1.92 ± 0.32 5.95 ± 0.29 9.86 ± 0.45 9.69 ± . a a c c A. salinestris AT  1.72 ± 0.27 2.09 ± 1.08 3.42 ± 0.21 2.22 ± . a a a a A. salinestris AT  1.34 ± 0.09 6.09 ± 0.43 15.38 ± 0.68 18.09 ± . a a a a A. salinestris AT  2.15 ± 0.08 3.41 ± 0.69 12.38 ± 0.37 19.47 ± . a a c c A. salinestris AT  1.90 ± 0.33 3.64 ± 0.30 5.14 ± 0.76 5.93 ± . a a b b A. salinestris AT  1.24 ± 0.76 3.32 ± 0.60 7.21 ± 0.40 10.62 ± .  a a c c A. salinestris AT  0.96 ± 0.55 1.82 ± 0.08 5.22 ± 1.28 6.25 ± . a a b b A. salinestris AT  1.45 ± 0.60 2.91 ± 0.43 8.23 ± 0.12 13.59 ± . a a c c A. salinestris AT 1.38 ± 0.13 3.20 ± 0.72 5.51 ± 0.15 6.58 ± . Auxin concentrations were determined aer , , , and  days of growth in liquid culture. Values are means of two replicates ± standard error (=2). Same letters in a column indicate no signicant di erences as determined by the DGC test ( = 0.05).

T : E ect of pure IAA solutions and Azotobacter inoculation on the number of seminal roots, and root colonization of -day-old wheat seedlings.

  Treatment IAA concentration g mL− Number of seminal roots per plant Root colonization (cfu root− ) c Water 4.33 ± . — b Low-IAA  4.69 ± . — a High-IAA  5.03 ± . — b  c A. salinestris AT . 4.68 ± . . ×  ± . × 104 b  b A. salinestris AT . 4.74 ± . .  ×  ± . × 105 a a A. chroococcum AT . 5.18 ± . . ×  ± . × 105 a  b A. chroococcum AT . 5.33 ± . . ×  ± . × 105 a a A. salinestris AT  . 5.39 ± . . ×  ± . × 106   Pregerminated seeds were treated with water (Control), IAA pure solutions of  g mL− (Low-IAA) and  g mL− (High-IAA), or were inoculated with A. salinestris strains (AT , AT , and AT ) or A. chroococcum strains (AT and AT). Values are means ± standard error of two independent experiments with three replicates (=6). Di erent letters in a column indicate signicant di erences between means as determined by the DGC test ( = 0.05).

A. salinestris AT and A. chroccoccum AT strains pre- observed when strains AT and AT were compared. −1 sented intermediate levels ( . mmol C2H4 mg protein Striking results were obtained with A. chroccoccum strain −1 −1 AT, whose production of the three phytohormones was  h ), and the lowest values ( mmol C2H4 mg protein  h−1) were found inA. salinestris AT and AT strains always in intermediate levels (Figures (a), (b), and(c) ). (Figure (d)). A strong agreement was observed between auxin production A. salinestris AT produced the highest level of IAA measured by the Salkowski reagent method and IAA pro- ( . gmL−1), the lowest level of GA (. gmL−1), and duction determined by GC-MS-SIM, excepting AT strain 3 (Table  and Figure (a)). an intermediate value of Z (. gmL−1). By contrast, A. salinestris AT and AT showed the lowest levels of −1 IAA production (.–. gmL ) and the highest levels .. E ects of Azotobacter Inoculation and IAA Pure Solutions −1 of GA3 production (. gmL ). ese two strains, how- on Root Morphology of Wheat Seedlings. Five strains were ever, di ered in their Z synthesis: while AT was one of used for inoculation assays, where all of them induced a the largest Z producers (. gmL−1), AT exhibited the signicant increase (on average  %) in the number of semi- lowest production (. gmL−1). Similar tendencies were nal roots of wheat seedlings (Table ). e greatest increase e Scientic World Journal

25 1.0

20 a a a )

b )

−1 b 15 b −1 c

g mL g 0.5  gmL c  10 c ( 3 d IAA ( IAA GA 5 d d

0 0 AT18 AT37 AT42 AT19 AT25 AT31 AT18 AT37 AT42 AT19 AT25 AT31 (a) (b)

1.5 20 ) −1

a h a 24 a 15 a −1

) 1.0 b b −1 b

gmL 10  mg protein mg (

c 4 b Z b H

0.5 2

Nitrogenase activity Nitrogenase 5 c c (mmol C (mmol 0 0 AT18 AT37 AT42 AT19 AT25 AT31 AT18 AT37 AT42 AT19 AT25 AT31 (c) (d)

F : Phytohormone production and nitrogenase activity by the selected Azotobacter strains. (a) Indole--acetic acid (IAA) production;

(b) gibberellic acid (GA3) production; (c) zeatin (Z) production, and (d) nitrogenase activity. IAA and GA3 were identied and quantied by gas chromatography-mass spectrometry, Z was identied and quantied by HPLC-UV, and nitrogenase activity (acetylene-ethylene reduction) was determined by gas chromatography. Bars are means of three replicates. e same letters indicate no signicant di erences between means as determined by the DGC test ( = 0.05).

of roots by Azotobacter. For instance,A. salinestris AT and A. chroococcum AT showed similar values of root colonization (on average . × 5 cfu root−1), but the latter was the one showing the largest positive e ect on the number of seminal roots. Maybe, a more direct relationship could be established between the stimulation of this feature and the Water Low-IAA High-IAA AT18 AT19 relative amount of phytohormones excreted by the inoculated F : E ect of IAA pure solutions and cell-free cultures of Azotobacter strains (Figures (a) and (c)). A. salinestris treatments on root morphology of -day-old wheat e e ect of cell-free culture and IAA-pure solution seedlings. Root tips of wheat seedlings treated with solutions of treatments on the number of root hairs was evaluated on  g mL−1 and  g mL−1 of IAA (low-IAA and high-IAA, resp.) -day-old wheat seedlings. Treatments with cell-free culture and cell-free cultures of low- (AT ) and high- (AT ) auxin- resulted in a stimulation of root hair number (Figure ) when producing Azotobacter strains. compared with control. A higher e ect was observed with cell-free culture of AT strain than that of AT strain. is e ect could be mimicked replacing cell-free culture of AT in the number of seminal roots (%) was obtained when strain by the high-IAA ( gmL−1) pure solution (Figure ). treated with the high IAA-pure solution and inoculating with In contrast, both cell-free cultures of AT strain and low- the three high-IAA-producing strains (A. chroococcum AT IAA pure solution treatments had a lesser e ect on root hair and AT and A. salinestris AT ). e results of bacterial production, compared with the AT cell-free culture or the inoculation did not seem to be related to the colonization high-IAA solution (Figure ).  e Scientic World Journal

4. Discussion A. beijerinckii, A. chroococcum, A. paspali, and A. vinelandii has been reported by researchers since   [], as far as we e genotypic characterization of Azotobacter native isolates are concerned, this is the rst report of in vitro phytohormone allowed us to identify three Azotobacter species and several production by A. salinestris strains. strains that showed a remarkable diversity. Among the  Our results suggest that these isolated Azotobacter strains strains isolated from  locations in Argentina, including both have the potential capacity to promote plant growth directly, agricultural and non-agricultural soils, A. chroococcum and through physiological mechanisms such as phytohormone A. salinestris were the species showing the highest frequency production, in addition to biological nitrogen xation and ( % and %, resp.). is result is in agreement with other siderophore production. e observed changes in root mor- studies that reported A. chroococcum as the most common phology aer inoculation with Azotobacter or cell-free cul- species isolated from soils [, , ]. However, considering ture treatment seem to be directly related to the capacity that less than a half soil samples contained azotobacteria ( of each strain to synthesize IAA. In previous studies, it was samples from a total of  analyzed soils samples), Azotobac- shown that root hairs and seminal roots can be a ected by ter species do not seem to be frequently found in Argentinean IAA concentration [ , ]. Nonetheless, it is well known that soils. Also, the isolation of Azotobacter was interestingly more other phytohormones are involved in regulating plant growth recurrent in non-agricultural than in agricultural soil samples and development. GA3 and Z, for instance, have also been ( % versus %, resp.). Even though there are no similar previously associated with the stimulation of many aspects previous reports in the literature, these results may indicate of plant growth [] but, despite this, it is known that plant a decrease of azotobacteria in anthropogenically disturbed hormones rarely function alone, and, even in cases in which soils. Hence, the application of biofertilizers with Azotobacter responses appear to be directly linked to the application of a might make up, at least partially, the loss of this benecial single hormone, these responses can also be a consequence of bacterial genus in agricultural systems. other endogenous hormones that are present in plant tissues e identication of A. salinestris and A. armeniacus in []. Argentinean soil samples was a surprising result because, until now, few reports have mentioned the isolation of these 5. Conclusions species. e presence of A. salinestris was reported in soils of western Canada [], while A. armeniacus was reported e genotyping of azotobacterial isolates by the combined in soils of Armenia [ ]. Although the isolation frequency of analysis of ARDRA and rep-PCR and the screening of isolates both species from soil seems to be low, our results suggest that based on their in vitro traits for potential plant growth they might have a more worldwide distribution than thought. promoting activity were useful tools for their taxonomic Another surprising result was that no A. vinelandii strain was classication and phenotypic characterization. is survey, isolated in our study, although this species has been reported embracing di erent regions of Argentina, allowed us to have as a common soil inhabitant [ ,  ]. Discrepancies found a rst approach to the presence of this bacterial genus in between our study and earlier reports may be attributed, at soils. Evaluation of plant growth-promoting traits in bacterial least in part, to the identication methodology used. Some strains is a very important task as criteria for strain selection misclassications might have occurred in the past [ ] due for biofertilizer formulations. As biofertilizers are a complex to the scarcity of genotypic characterizations of Azotobacter resulting from bacteria and their metabolites excreted to the isolates. In addition, the sources from where the isolates were growing medium, it becomes relevant to evaluate every con- withdrawn could also explain these di erences: in many stituent of a biofertilizer before considering it as a potential previous studies, Azotobacter strains were isolated from rhi- candidate for eld application. us, our results constitute an zospheric soil, while in this study, the isolates were obtained important technological contribution to Azotobacter strain from bulk soil, a fraction not directly inuenced by root selection for biofertilizer formulations that would help to activity. Our results reveal the wide tolerance of Azotobacter implement a more sustainable agriculture through decreasing genus to di erent climate conditions, types of soil, and soil the use of agrochemicals. characteristics such as organic matter content, pH values, and phosphorous concentrations. Conflict of Interests IAA and GA3 production in our collection of Azotobacter strains was higher than that reported for a phyllospheric A. e authors declare that there is no conict of interests regarding the publication of this paper. chroococcum strain REN2 [ ]. Conversely, other Azotobacter strains, isolated from rhizospheric soil in India, reached the same IAA production levels than our high-IAA-producing Acknowledgments strains [ ]. Although all tested strains excreted phytohor- mones in chemical complex growing medium, the levels of e authors thank the Instituto Nacional de Tecnolog´ıa IAA, GA3, and Z production di ered among them. Interest- Agropecuaria (INTA), the Instituto de Investigaciones en ingly, IAA production showed high levels in almost all A. Biociencias Agr´ıcolas y Ambientales (INBA-CONICET/ chroococcum strains but variable levels in A. salinestris strains, UBA), and Catedra´ de Microbiolog´ıa Agr´ıcola, Facultad de agreeing with its higher intraspecic diversity revealed by Agronom´ıa, Universidad de Buenos Aires, for their support rep-PCR. Even though the production of phytohormones by to carry out this research. e Scientic World Journal 

References [ ] S. F. Altschul, T. L. Madden, A. A. Scha er¨ et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database [] C. Kennedy, P.Rudnick, M. MacDonald, and T. Melton, “Genus search programs,” Nucleic Acids Research, vol. , no.  , pp. III: Azotobacter,” in Bergey’s Manual of Systematic Bacteriology.  –,  . e Proteobacteria, Part B, the ,G.M. [ ] S. Perez-Miranda,´ N. Cabirol, R. George-Tellez,´ L. S. Zamudio- Garrity, Ed., vol. , pp.  –, Springer, New York, NY, USA, Rivera, and F. J. Fernandez,´ “O-CAS, a fast and universal nd edition, . method for siderophore detection,” Journal of Microbiological [] L. Aquilanti, F. Favilli, and F. Clementi, “Comparison of dif- Methods, vol. , no. , pp.  –,  . ferent strategies for isolation and preliminary identication of [ ] R. I. Pikovskaya, “Mobilization of phosphorus in soil in con- Azotobacter from soil samples,” Soil Biology and Biochemistry, nection with vital activity of some microbial species,” Microbi- vol.  , no. , pp.  – , . ologiya, vol.  , pp.  – ,   . [] A. Hussain, M. Arshad, A. Hussain, and E. Hussain, “Response [ ] C. S. Nautiyal, “An ecient microbiological of maize (Zea mays) toAzotobacter inoculation under fertilized for screening phosphate solubilizing microorganisms,” FEMS and unfertilized conditions,” Biology and Fertility of Soils, vol. , Microbiology Letters, vol.  , no. , pp.  – ,  . no. -, pp. – ,  . [] E. Glickmann and Y. Dessaux, “A critical examination of the [] R. Behl, N. Narula, M. Vasudeva, A. Sato, T. Shinano, and M. specicity of the Salkowski reagent for indolic compounds pro- Osaki, “Harnessing wheat genotype X Azotobacter strain inter- duced by phytopathogenic bacteria,” Applied and Environmental actions for sustainable wheat production in semi arid tropics,” Microbiology, vol. , no. , pp. – ,  . Tropics, vol. , pp. –,  . [] D. Perrig, M. L. Boiero, O. A. Masciarelli et al., “Plant- [] R. Kizilkaya, “Yield response and nitrogen concentrations of growth-promoting compounds produced by two agronomically spring wheat (Triticum aestivum) inoculated withAzotobacter important strains of Azospirillum brasilense, and implications chroococcum strains,” Ecological Engineering, vol. , no. , pp. for inoculant formulation,” Applied Microbiology and Biotech- – ,  . nology, vol. , no. , pp. –,  . [ ] I. R. Kennedy, A. T. M. A. Choudhury, and M. L. Kecskes,´ “Non- [] J. A. Di Rienzo, A. W.Guzman,´ and F. Casanoves, “A multiple- symbiotic bacterial diazotrophs in crop-farming systems: can comparisons method based on the distribution of the root node their potential for plant growth promotion be better exploited?” distance of a binary tree,” Journal of Agricultural, Biological, and Soil Biology and Biochemistry, vol.  , no. , pp.  –, . Environmental Statistics, vol. , no. , pp.  –, . [ ] V. Kumar and N. Narula, “Solubilization of inorganic phos- [] J. A. Di Rienzo, F. Casanoves, M. G. Balzarini, L. Gonzalez, phates and growth emergence of wheat as a ected by Azotobac- M. Tablada, and C. W. Robledo, InfoStat Version´   , Grupo ter chroococcum mutants,” Biology and Fertility of Soils, vol.  , InfoStat, FCA, Universidad Nacional de Cordoba,´ Cordoba,´ no. , pp. –,  . Argentina, http://www.infostat.com.ar/. [ ] M. Khalid, Z. A. Zahir, A. Waseem, and M. Arshad, “Azotobac- [] L. Aquilanti, I. Mannazzu, R. Papa, L. Cavalca, and F. Clementi, ter and L-tryptophan application for improving wheat yield,” “Amplied ribosomal DNA restriction analysis for the charac- Pakistan Journal of Biological Science, vol. , pp.  – ,  . terization of Azotobacteraceae: a contribution to the study of [ ] B. R. Pati, S. Sengupta, and A. K. Chandra, “Impact of selected these free-living nitrogen-xing bacteria,” Journal of Microbio- phyllospheric diazotrophs on the growth of wheat seedlings and logical Methods, vol.  , no. , pp.  – , . assay of the growth substances produced by the diazotrophs,” [] W. J. Page and S. Shivprasad, “Azotobacter salinestris sp. Microbiological Research, vol. , no. , pp. – ,  . nov., a sodium-dependent, microaerophilic, and aeroadaptive [] Z. A. Zahir, H. N. Asghar, M. J. Akhtar, and M. Arshad, “Pre- nitrogen-xing bacterium,” International Journal of Systematic cursor (L-tryptophan)-inoculum (Azotobacter) interaction for Bacteriology, vol. , no. , pp.  – ,  . improving yields and nitrogen uptake of maize,” Journal of Plant [ ] J. P. ompson and V. B. D. Skerman, Azotobacteraceae: e Nutrition, vol.  , no. , pp. –  , . and Ecology of the Aerobic Nitrogen-Fixing Bacteria, [] A. Boraste, K. K. Vamsi, A. Jhadav et al., “Biofertilizers: a novel J. P.ompson and V.B. D. Skerman, Ed., Academic Press,  . tool for agriculture,” International Journal of Microbiology [ ] D. Farajzadeh, B. Yakhchali, N. Aliasgharzad, N. Sokhandan- Research, vol. , pp. –,  . Bashir, and M. Farajzadeh, “Plant growth promoting character- [] D. L. Sparks, A. L. Page, P.A. Helmke et al., “Chemical methods,” ization of indigenous Azotobacteria isolated from soils in Iran,” in Methods of Soil Analysis, vol.   of ASA-Soil Science Society Current Microbiology, vol. , no. , pp.  –, . of America Book Series, American Society of Agronomy,  . [ ] L. Cavaglieri, J. Orlando, and M. Etcheverry, “In vitro inuence [] J. Versalovic, M. Schneider, F. J. de Bruijn, and J. R. Lupski, of bacterial mixtures on Fusarium verticillioides growth and “Genomic ngerprinting of bacteria using repetitive sequence- fumonisin B production: e ect of seeds treatment on maize based polymerase chain reaction,” Methods in Molecular and root colonization,” Letters in Applied Microbiology, vol. , no. Cellular Biology, vol. , no. , pp. –,  . , pp.  – , . [] M. S. Montecchia, N. L. Kerber, N. L. Pucheu, A. Perticari, [ ] F. Ahmad, I. Ahmad, and M. S. Khan, “Screening of free-living and A. F. Garc´ıa, “Analysis of genomic diversity among pho- rhizospheric bacteria for their multiple plant growth promoting tosynthetic stem-nodulating rhizobial strains from Northeast activities,” Microbiological Research, vol.  , no. , pp.  – , Argentina,” Systematic and Applied Microbiology, vol. , no. ,  . pp. –, . [] K. Kukreja, S. Suneja, S. Goyal, and N. Narula, “Phytohormone [] J. P. W. Young, H. L. Downer, and B. D. Eardly, “Phylogeny production by Azotobacter—a review,” Agricultural Reviews, of the phototrophic Rhizobium strain BTAi by polymerase vol. , pp. – , . chain reaction-based sequencing of a  S rRNA gene segment,” [] S. Spaepen, S. Dobbelaere, A. Croonenborghs, and J. Vander- Journal of Bacteriology, vol.  , no. , pp.  – ,  . leyden, “E ects of Azospirillum brasilense indole--acetic acid  e Scientic World Journal

production on inoculated wheat plants,”Plant and Soil, vol. , no. -, pp. –,  . [] L. Taiz and E. Zeiger, “e biological roles of cytokinins,” in Cytokinins. Plant Physiology, L. Taiz and E. Zeiger, Eds., chapter , pp. – , Sinauer Associates, Sunderland, Mass, USA,  .