Chapter 19

Phylogenetic Analysis of AzospirillumSpecies Isolated from the Rhizosphereof Field-Grown Wheat Based onGenetic and Phenotypic Features

Vezyri Eleni, Venieraki Anastasia, Dimou Maria, ChatzipavlidisIordanis, Tampakaki Anastasia, and Panagiotis KatinakisLaboratory of General and Agricultural Microbiology, Department of AgriculturalBiotechnology, Agricultural University of Athens, Greece

19.1 INTRODUCTION

The rhizosphere is a dynamic environment, wherenumerous interactions among plants and microorgan-isms take place. Root activities provide a nutrient-richenvironment for the microorganisms for their growth.Microorganisms inhabiting the root rhizosphere canbe grouped into two general categories, those havingbeneficial effects to plants and those having detrimentalones. In the former group, the bacterial populationsknown as plant growth–promoting rhizobacteria are ableto promote root and plant growth and in some cases arecapable of reducing the damage caused by soil-bornediseases (Fibach-Paldi et al., 2012; see Chapters 53, 54).

The genus Azospirillum encompasses free-living,nitrogen-fixing, plant growth–promoting rhizobacteriathat can influence growth and yield of many agronomi-cally important crops including rice and wheat (Bashanand De-Bashan, 2010). The plant growth activity ofAzospirillum is mainly attributed to the secretion ofauxin and production of nitrous oxide (NO) rather to thenitrogen-fixing ability of the bacteria. Auxin promotesplant root elongation, which results in more efficientuptake of nutrients (Steenhoudt and Vanderleyden, 2000;Bashan and De-Bashan, 2010).

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

To date, at least 16 species of the Azospirillum genushave been described; however, more attention has beengiven to the investigation of Azospirillum brasilense andAzospirillum lipoferum (Lin et al., 2011; Pothier et al.,2008). Complete genome sequences of four members ofAzospirillum species, A. brasilense Sp245 (Wisniewski-Dye et al., 2011), A. lipoferum 4B (Wisniewski-Dye et al.,2011), Azospirillum sp. B510 (Kaneko et al., 2010), andAzospirillum amazonense (Sant’Anna et al., 2011), areavailable in databases so far.

Members of Azospirillum genus exhibit a broadecological distribution and are found in many soiltypes in different geographical regions. In agriculturalecosystems, the Azospirillum population was foundin the bulk soil but is much more abundant in therhizosphere systems (Bashan, 1999). Cultivated soilsworldwide are becoming more saline due to irrigationpractices and excessive fertilization. However, increasedsalinity of soils appears to negatively affect the diversityof indigenous Azospirillum species (Saleena et al.,2002) and salinity stress (300 mM NaCl) inhibits thegrowth and nitrogen fixation ability of A. brasilense(Chowdhury et al., 2007). Inoculation of wheat seedlingsgrown under high NaCl concentration with A. lipoferumpartially alleviated the deleterious effects of high

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204 Chapter 19 Phylogenetic Analysis of Azospirillum Species Isolated from the Rhizosphere

salinity (Bacilio et al., 2004). Similarly, inoculationwith A. brasilense contributed to the protection of wheatseedlings grown under water stress (Pereyra et al., 2006).Genetically engineered A. brasilense producing elevatedlevels of trehalose exhibited more salinity resistancethan the wild-type strain and significantly enhanced thesurvival of maize growing under drought stress. It alsosignificantly increased biomass and leaf and root lengthof the plants (Rodriguez-Salazar et al., 2009).

Motility is a major taxonomic feature of the genusAzospirillum (Tarrand et al., 1978). Motility and chemo-taxis towards metabolites produced by crop plants are con-sidered as important factors for efficient root colonization(Bashan, 1999; see Chapter 17). Migration of A. brasilenseCd through the soil towards the wheat roots was charac-terized by a simultaneous migration of nearly all bacteriaand it was faster towards wheat seedlings than towardsnonplanted soil (Bashan, 1986). Motility within the genusAzospirillum is achieved via the motion of flagella (Wilsonand Beveridge, 1993). Azospirillum brasilense displaysmixed flagellation: possessing a single polar flagellum andmultiple lateral flagella. The polar flagellum is responsiblefor the swimming motility, while the latter for swarmingmotility (Hall and Krieg, 1983; Moens et al. 1995; Sche-lud’ko et al., 1998). Nonflagellated mutants as well as amutant strain impaired in chemotactic response showedreduced colonization of the root system suggesting thatactive movement of Azospirillum is important for theinitiation of root colonization (Vande Broek et al. 1998).

Swarm motility (collective surface movement) hasbeen described in both Gram-positive and Gram-negativebacteria, including Proteus, Pseudomonas, Azospirillum,Salmonella, Serratia, Vibrio, Clostridium, and Bacillus(Harshey, 2003; Kearns, 2010). The swarming phenotypeis widespread among Azospirillum species and swarmingproficiency appears to vary among strains of the sameAzospirillum species (Hall and Krieg, 1983; Baldaniet al., 1986): a more vigorous swarming phenotypewas observed in A. brasilense Sp245 compared toA. brasilense Sp7 (Baldani et al., 1986). Swarmingproficiency of A. brasilense Sp245 is stimulated byenvironmental factors such as wheat seedling exudates(Borisov et al., 2009), nitrogen source, and oxygentension (Schelud’ko et al., 2009). A limited number ofgenes involved in Azospirillum species swarming motilityhave been characterized so far (Moens et al. 1995;Carreno-Lopez et al., 2009).

Recently, we have isolated numerous nitrogen-fixingstrains from the rhizosphere of cereals and phylogeneticanalysis places them in the genus of Azospirillum andPseudomonas. The majority of the Azospirillum isolatesaffiliated, according to the 16S rRNA gene analysis, withA. brasilense as the closest valid described species while afew were placed with Azospirillum zeae (Venieraki et al.,

2011a, Venieraki et al., 2011b). Some of the latterisolates, however, exhibited a dissimilarity of their 16SrRNA gene nucleotide sequences exceeding the recentlyset upper limit of 1.3% in 16S rRNA sequences whichis used for species discrimination (Stackebrandt andEbers, 2006). In the present study, we assessed sequencediversity of the internal transcribed spacer (ITS) regionsin conjunction with 16S rRNA sequences to enhance thephylogenetic resolution of these isolates. Furthermore,the repetitive element palindromic (Rep)-PCR finger-printing approach was employed in order to compare thegenotypes of our isolates with those of type strain A. zeaeN7. Their swarming motility in response to salinity andtemperature was also investigated.

19.2 METHODS

19.2.1 Bacterial Strains andCulture ConditionsThe isolation of bacterial strains (Gr1, Gr2, Gr24,Gr31, Gr35, and Gr60) used in this study has beendescribed previously (Venieraki et al., 2011a, 2011b).Azospirillum zeae N7 (A. zeae; Mehnaz, Weselowski,and Lazarovits (2007) VP) LMG23989 and A. lipoferumsp59b were obtained from the Belgian CoordinatedCollections of Micro-organisms, Laboratory for Micro-biology of the Faculty of Sciences of Ghent University(BCCM/LMG). All Azospirillum strains were grown inLB/MC (1% Bacto Tryptone, 0.5% Yeast extracts, 1%NaCl, 2.5 mM MgSO4, and 2.5 mM CaCl2) at 30 ◦C.

19.2.2 Swarming MotilityThe swarming motility was assessed on swarm plates withLB/MC containing 0.6% agar (Fluka 05039). To assess theeffect of NaCl concentration and temperature on swarm-ing, migration strains were grown on 0.6% agar LB/MCsupplemented with 1%, 3%, or 5% NaCl and incubated at30 ◦C, 37 ◦C, or 42 ◦C for 18h. The inoculation of seedswas carried out by culturing the isolates in liquid LB/MCand placing 3 ml of each culture on the centre of the swarmplate.

19.2.3 DNA Fingerprinting AnalysisRep-PCR DNA fingerprinting was performed using therep primer set according to the protocol described byRademaker et al. (2000) to examine the relatedness of thestrains (Ishii and Sadowsky, 2009). Amplified DNA frag-ments were separated by electrophoresis in 2% agarosegel at 70 V for 4 h.

19.3 Results 205

19.2.4 DNA Extraction, ITS1Amplification, and SequencingAll the studied strains were grown in LB/MC overnightand DNA was extracted using the GenElute BacterialGenomic DNA Kit according to the manufacturer’sinstructions for Gram-negative bacteria (Sigma-Aldrich).DNA concentrations were determined using a Nan-odrop ND-1000 spectrophotometer. The ITS1 regionwas amplified using a pair of oligonucleotide primers,A1F (Rrn16S-f: 5′-GAAGTCGTAACAAGG) and A2R(Rrn23S-R: CAAGGCATCCACCGT). The PCR assayconditions and cloning of the amplicons was carriedout essentially as described by Venieraki et al. (2011a).Sequences were edited using Seqbuilder (Lasergene),and MEGALIGN 7.06 software (Lasergene) was usedto perform multiple alignments with other ITS1 or ITS2sequences of Azospirillum reported in the NCBI Data-Bank. Phylogenetic trees based on nucleotide sequencesof ITS1 and ITS2 fragments were constructed withMEGA 5.0 using the neighbor-joining algorithm (1000bootstrap replication) (Tamura et al., 2011). The tRNAsequences were identified using the program tRNAscan(Lowe and Eddy, 1997).

19.2.5 Nucleotide SequenceAccession NumbersThe nucleotide sequences determined in this study havebeen deposited in the GenBank database under accessionnumbers HE717080-HE717995.

19.3 RESULTS

19.3.1 Phylogenetic Affiliation ofthe Isolates Based on 16S rRNA andITS1 SequencesA collection of six isolates, five isolated from the rhizo-sphere of wheat and one isolated from the rhizosphereof oat were previously identified as A. zeae based ontheir 16S rRNA gene sequences (Venieraki et al., 2011a,2011b). Pairwise comparisons of the 16S rRNA genesequences revealed sequence similarities ranging from97.4% to 99.8% when the six isolates were comparedwith each other. In addition, comparison of the sixisolates with reference strains of Azospirillum type strainsdisplayed sequence similarities to the type strains of A.zeae N7, Azospirillum melinis, Azospirillum oryzae, andAzospirillum sp. B510 with similarity values ranging from97.4% to 99.0%, 97.6% to 98.7%, 98.3% to 99%, and97.6% to 98.6%, respectively. To gain an insight into thephylogenetic relationships of the isolates, a phylogenetic

tree was constructed including 16S rRNA gene sequencesof closely related Azospirillum species retrieved fromGenBank. An inspection of the phylogenetic tree revealedthat the isolates represented novel sublines within thegenus Azospirillum: The closest relative to Gr60 is A.oryzae, Gr24 and Gr31 are clustered adjacent to A. zeaeN7 while Gr35 is clustered adjacent to A. lipoferum,and isolates Gr1 and Gr2 form a separate branch withinAzospirillum (Fig. 19.1a).

The PCR-amplified ITS1 products were representedby two distinct bands (one with a low molecular weight,designated as ITS1S and band with larger molecularweight designated as ITS1L) on agarose gels for allisolates including the type strain A. zeae N7. The relativeintensities of the ITS1 bands significantly differed: theITS1L band being more intense than this ITS1S band(Data not shown). Analyses of the nucleotide sequencesof both ITS1 region indicated that all ITS1L regions con-tained two deduced tRNA genes, tRNAAla and tRNAIle,while the ITS1S regions did not contain tRNA genes.The phylogenetic tree inferred from ITS1L and ITS1Ssequences demonstrated that the isolates are grouped in amanner further strengthening the indice that the isolatesrepresent novel sublines within the genus Azospirillum(Fig. 19.1b and c).

19.3.2 Genotypic Analysis of theIsolates by Rep-PCRThe Rep-PCR is a versatile and efficient method for thefingerprinting of bacterial isolates (Rademaker et al.,2000; Ishii and Sadowsky, 2009). It has been reportedthat the Rep-PCR approach generates unique PCR finger-prints for each isolate of Azospirillum facilitating theirdiscrimination at the strain level (Ishii et al., 2011; Tejeraet al., 2005). To further examine the genetic relatednessof the six isolates, the Rep-PCR analysis was carriedout. When Rep primers were used for amplificationof DNA from the six tested isolates and the referencestrain A. zeae N7, several weak and strong bands ofPCR products, ranging from about 0.3 kb–23.5 kb, weredetected (Fig. 19.2). A visual inspection revealed thatthe banding pattern of all isolates was different fromthat observed for A. zeae. Furthermore, each isolatefrom the wheat and oat rhizosphere exhibited distinctbanding patterns of PCR products, with exception ofstrains Gr1 and Gr2, suggesting that these isolates couldbe considered as different strains. The Rep-PCR profilesof isolates Gr1 and Gr2 are quite similar but not identical(Fig. 19.2). Thus, we cannot rule out the possibility thatthese isolates might be clones of the same strain.

206 Chapter 19 Phylogenetic Analysis of Azospirillum Species Isolated from the Rhizosphere

(a)

(c)

(b)

Figure 19.1 Phylogenetic trees of 16S rDNA(a), ITS1S (b), and ITS1L (c) of six isolates(Gr1, Gr2, Gr24, Gr31, Gr35, and Gr60) obtainedfrom the rhizosphere soil of wheat and oat,Azospirillum zeae N7 and related sequencesretrieved from NCBI. Numbers shown at nodesindicate bootstrap values (percentage of 1000replicates). The bar scale indicates the rate ofsubstitution per nucleotide position. Sequenceaccession numbers are given in parentheses.

19.3.3 Swarming Motility of theSix Isolates Under DifferentEnvironmental ConditionsTo gain an insight into the swarming motility competenceof the test strains and to assess the effect of salinity and

temperature on their swarming mobility, swarm assayswere performed under various salinity and/or temperatureconditions. The swarming motility was assessed on swarmplates containing LB/MC with 0.6% agar, supplementedwith either 3% or 5% NaCl or just LB/MC and incubated

19.4 Discussion 207

Figure 19.2 Molecular fingerprinting banding patterns with Repprimers of six isolates (Gr1, Gr2, Gr24, Gr31, Gr35, and Gr60) andAzospirillum zeae N7.

at 30 ◦C, 37 ◦C, or 42 ◦C. A “motility zone” formed bymigrating bacteria was observed in all strains tested, withthe exception of strain Gr60, at all tested temperatures andsalinities. No growth of strain Gr60 was observed whenit was cultured with 5% NaCl irrespective of the growthtemperature. The swarming migration velocity appears tovary significantly among the under study strains. StrainsGr1, Gr31, Gr60, and A. zeae N7 (henceforth referred asweak swarmers) exhibited a motility zone with a diameterranging from 4 to 12 mm while strains Gr2, Gr24, andGr35 (henceforth referred as vigorous swarmers) showeda motility zone ranging from 8 to 85 mm depending on theincubation temperature and/or salinity levels and bacterialstrain (Table 19.1). The swarm migration of the majorityof the strains was inhibited by the increased NaCl concen-tration in the growth medium while elevated incubationtemperature appeared to enhance the swarm migration ofstrains Gr2, Gr24, and Gr35 regardless the salinity levels(Table 19.1). Representative images of swarm motility ofthe vigorous swarmer strain Gr35 is shown in Figure 19.3.It is evident that increase of salt concentration in thegrowth medium resulted in a significant decrease in theratio of swarm halo (Fig. 19.3A, D, and G), whereas

increase of incubation temperature resulted in an increasein the diameter of the swarm halo of strain Gr35 underhigh salt (3% NaCl concentration) conditions (Fig. 19.3D,E, and F).

19.4 DISCUSSION

In the present study, a total of six isolates from the rhizo-sphere of field-grown wheat and oat plants collected fromvarious regions of Greece were characterized by means ofsequence analysis of 16S rRNA, ITS1 regions, Rep-PCRfingerprinting, and phenotypic characterization based ontheir swarming capacity.

In a recent study, the recommended 16S rRNA genesequence similarity among different bacterial species wasincreased from 97% to 98.7% in order to facilitate tax-onomic studies (Stackebrandt and Ebers, 2006). Further-more, ITS1 regions exhibit a high variation in length andsequence and have been suggested to be well suited fortyping and identification of bacteria at both the speciesand the strain level (Gurtler and Stanisich, 1996).

Our data demonstrate that all tested strains harbortwo types of ITS1, ITS1L and ITS1S. The differencesin intensities between ITS1L and ITS1S PCR ampliconscould reflect the copy number of rRNA operons (rrns)containing the respective ITS1. Nine rrns have beenidentified within each of the sequenced genomes of A.lipoferum 4B and Azospirillum sp.B510 (Kaneko et al.,2010; Wisniewski-Dye et al., 2011). Both genomes carrytwo ITS1 types varying in length: the larger ITS1 (ITS1L)exhibits a length of about 600 bp and the shorter (ITS1S)a size of about 290 bp. In both genomes, the ITS1Stype is found in only one out of nine rrns while theIST1L type is found in the remaining ones. The presenceof two types of ITS1, with variable lengths, have alsobeen reported in A. brasilense Sp245 and A. lipoferumCRT1 (Baudoin et al., 2010). However, a BlastN analysisusing the published sequences of ITS1S (AY685928) asa probe revealed the absence of this sequence from thesequenced genome of A. brasilense Sp245 kept in Lyon.This discrepancy may be attributed to missing DNAsequences during the sequencing of the genome.

Our data inferred from the phylogenetic trees of 16SrRNA gene as well as ITS1S and ITS1L sequences sug-gested that strain Gr60 is affiliated to A. oryzae, strainsGr24 and Gr31 are affiliated to A. zeae, strain Gr35 isaffiliated to A. lipoferum, and strains Gr1 and Gr2 maybelong to a new Azospirillum species.

Our phylogenetic data suggest that strains Gr24and Gr31 isolated from the rhizosphere of Triticumturgidum var. durum and Triticum aestivum, respectively,are closely related and may be affiliated to A. zeae N7.However, the Rep-PCR patterns of these strains are

208 Chapter 19 Phylogenetic Analysis of Azospirillum Species Isolated from the Rhizosphere

Table 19.1 Effect of NaCl and/or temperature on swarming migration of the six isolates

NaCl Treatment—Colony Diameter (mm)

1% NaCl 3% NaCl 5% NaCl

Strain/◦C 30 37 42 30 37 42 30 3 4

Gr1 9 ± 2 12 ± 3 10 ± 2 6 ± 2 8 ± 2 5 ± 2 5 ± 1 5 ± 2 4 ± 1Gr2 70 ± 8 85 ± 4 85 ± 5 30 ± 5 60 ± 9 20 ± 4 10 ± 4 18 ± 3 15 ± 3Gr24 30 ± 6 5 ± 1 70 ± 9 6 ± 1 20 ± 5 55 ± 7 5 ± 1 7 ± 4 30 ± 5Gr31 6 ± 1 6 ± 2 6 ± 1 5 ± 1 5 ± 2 5 ± 1 4 ± 2 4 ± 1 4 ± 1Gr35 85 ± 5 70 ± 9 85 ± 5 20 ± 4 30 ± 5 85 ± 9 5 ± 1 7 ± 2 8 ± 2Gr60 6 ± 2 6 ± 1 6 ± 2 5 ± 2 4 ± 2 4 ± 1 — — —Azospirillum zeae N7 6 ± 1 6 ± 2 6 ± 1 5 ± 1 5 ± 1 5 ± 2 4 ± 1 4 ± 1 4 ± 1

Diameter of bacterial displacement of the six isolates (Gr1, Gr2, Gr24, Gr31, Gr35, and Gr60) on swarm plates after incubation for 18 h assessed asdescribed in Methods section. Data represent the means ± standard deviation from three experiments with three replicate plates per treatment in eachexperiment.

Figure 19.3 Swarming motility patterns of strain Gr35 at differentNaCl concentrations and/or temperatures. Swarm plates (LB/MCwith 0.6% agar) (A, B, and C) containing 3% (D, E, and F), and 5%NaCl (G, H, and I) were inoculated with exponentially growing cellsof strain Gr2 and incubated for 18 h at 30 ◦C ( A, D, and G), 37 ◦C(B, E, and H), and 42 ◦C (C, F, and I). The results are representativeof those from three independent experiments.

quite different to those generated using the referencestrain A. zeae N7. In contrast, similar Rep-PCR patternswere observed among A. brasilense ATCC 21145 and A.brasilense strains isolated from sugarcane rhizosphere(Tejera et al., 2005). A recent study demonstrated that phe-notypic variants of A. brasilense ATCC 29145 collectedafter prolonged starvation or reisolated after colonizationof maize roots showed either quite different Rep-PCRpatterns and/or different plasmid profiles (Lerner et al.,2010). The authors suggested that rearrangements may

take place within a short time under certain environmentalconditions within Azospirillum species and they mayaccount for the different Rep-PCR patterns. It is worthnoting that Azospirillum species are prone to genomerearrangements compared to other proteobacteria suchas Sinorhizobium species (Wisniewski-Dye et al., 2011).Though the Rep-PCR profiles have been proven sufficientin grouping Sinorhizobium strains (Zribi et al., 2005), thismay not apply to Azospirillum. Taken together, the smalldifferences in Rep-PCR profiles observed among ourstrains affiliated to A. zeae may be the result of genomerearrangements. It is worth pointing out that irrigation ofwheat fields in Greece is based on ground water whichresults in accumulation of salts in soil. Therefore, it isplausible to suggest that wheat rhizosphere in Greecemay be colonized by Azospirillum populations adapted tohigh salt environments.

It has been reported that that active movement ofAzospirillum is important for the initiation of root colo-nization (Vande Broek et al., 1998). Furthermore, wheatroot colonization by A. brasilense swarming motilitymutants (SWa− Gri+) showed a lower capacity for wheatroot adsorption (Shelud’ko et al., 2010). In the presentstudy, we showed that five out of six isolates were ableto swarm in LB/MC or in LB/MC supplemented with3% NaCl or 5% irrespective of incubation temperature.Hence, it will be of interest to determine whetherswarming motility of our isolates in soils with increasingconcentration of NaCl affects colonization efficiency, inorder to select Azospirillum strains, which may be usefulas inoculants in salt-affected fields.

Our data also demonstrated that three out of sixAzospirillum species exhibited a vigorous surface migra-tion at the normal incubation temperature of 30 ◦C in agrowth medium (LB/MC) which contains relatively high

References 209

NaCl concentration (1%). The presence of these pheno-types among our isolates raises the question whether avigorous swarming phenotype may confer an advantagefor colonization of the rhizosphere. Flagella-drivenbacterial motility is required in the establishment of A.brasilense Sp7 colonization of wheat roots (Vande Broeket al., 1998). The involvement of swarming motilityin the colonization process has been reported duringcolonization of the alfalfa rhizosphere by P. fluorescenceF113 (Sanchez-Contreras et al., 2002). Recent studieshave also shown that endophytic colonization of rice rootswas influenced by the twitching motility of Azoarcus sp.BH72 (Bohm et al., 2007). Whether swarming plays arole in the Azospirillum root colonization is not clear yet(Shelud’ko et al., 2010).

It has been demonstrated that bacterial swarmingmotility is influenced by environmental conditions suchas high concentration of NaCl in the growth medium andtemperature of incubation. In Rhizobium leguminosarumsurface migration was observed at 22 ◦C but not at 30 ◦C(Matilla et al., 2007). Stimulation of swarming motilityat low temperature has been demonstrated in Serratiamarcescens (Lai et al., 2005) and Pseudomonas putidaKT2440 (Matilla et al., 2007).

On the other hand, Bacillus subtilis strain 3610showed an enhancement of swarming migration atelevated temperatures (Julkowska et al., 2004). IncreasingNaCl exerted an inhibitory effect on the swarmingmigration of Pseudomonas strains isolated from LakeBaikal (Soutourina et al., 2001). Photorhabdus temperata,on the other hand, required the presence of additionalNaCl or KCl in the growth medium in order to exhibit avigorous swarming phenotype (Michaels and Tisa, 2011).Analysis of the swarming motility capacity of the sixAzospirillum isolates at different NaCl concentrationsdemonstrated that swarming migration of vigorousswarmer isolates (Gr2, Gr24, and Gr35) was more sensi-tive to environmental conditions such as increasing NaClconcentration, whereas elevated temperature appearsto exert a stimulatory effect on swarming migrationregardless of the NaCl concentration. Interestingly, theinhibitory effect of NaCl on swarming motility of theseisolates was partially alleviated by elevated temperature(Fig. 19.3). Interestingly, the Rep-PCR profiles showedthat strains Gr1 and Gr2 shared over 80% of the visiblebands indicating their close genetic relatedness. However,strains Gr1 and Gr2 exhibited a strain-specific swarmingphenotype: strain Gr2 displays a vigorous swarmingphenotype compared to strain Gr1.

In conclusion, our data demonstrate that the pre-viously described A. zeae strains consist of differentAzospirillum species. In addition, our Azospirillum straincollection is grouped in weak and vigorous swarmers,with the swarming ability of the latter group being

profoundly affected by the environmental conditions.Further studies will shed light on the correlation, if itexists, between the swarming ability and colonizationefficiency of the strains tested. Finally, the presentfindings, taken together, indicate that a combined analysisbased on genetic and phenotypic features improves thediscrimination of strains belonging to Azospirillum genusand provide new insights into their phylogeny.

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