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Biology and Fertility of SoilsCooperating Journal of InternationalSociety of Soil Science ISSN 0178-2762 Biol Fertil SoilsDOI 10.1007/s00374-013-0854-y

The endophyte Enterobacter sp. FD17: amaize growth enhancer selected based onrigorous testing of plant beneficial traitsand colonization characteristics

Muhammad Naveed, Birgit Mitter,Sohail Yousaf, Milica Pastar,Muhammad Afzal & Angela Sessitsch

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ORIGINAL PAPER

The endophyte Enterobacter sp. FD17: a maize growthenhancer selected based on rigorous testing of plant beneficialtraits and colonization characteristics

Muhammad Naveed & Birgit Mitter & Sohail Yousaf &Milica Pastar & Muhammad Afzal & Angela Sessitsch

Received: 22 June 2013 /Revised: 25 August 2013 /Accepted: 27 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract With the aim to select powerful microbial strains tobe used for the enhancement of maize yield and resistance toabiotic and biotic stresses, we tested five endophytic bacterialstrains previously isolated frommaize roots. A range of differentlaboratory assays in regard to potential plant growth promotionwas performed and strains were further evaluated for improvinggrowth of five maize cultivars under axenic and natural soilconditions. Endophytic colonization was an additional compo-nent in our selection process as it is of high importance for aninoculant strain to efficiently colonize the plant environment. Allstrains had the potential to improvemaize seedling growth underaxenic conditions. Enterobacter sp. strain FD17 showed boththe highest growth-promoting activity under axenic conditionsas well as colonization capacity. FD17was therefore selected forfurther plant tests in a net house, in which two different maizecultivars were grown in large pots until ripening and subjected tooutdoor climatic conditions. Results showed that inoculationsignificantly increased plant biomass, number of leaves plant−1,leaf area, and grain yield up to 39 %, 14 %, 20 %, and 42 %,respectively, as compared to the un-inoculated control.Similarly, inoculation also improved the photochemical efficien-cy of photosystem II (PSII) of maize plant and reduced the timeneeded for flowering.We also confirmed that strain FD17 is ableto colonize the rhizosphere, roots and stems. Based on rigoroustesting, Enterobacter sp. strain FD17 showed the highest

potential to promote growth and health of maize grown undernatural conditions. This study suggested that in vitro plantgrowth-promoting traits and potential of maize seedling growthpromotion by bacterial endophytes could be used for the selec-tion of potential inoculant strains subjected for further testing asbio-inoculant under field conditions.

Keywords Enterobacter sp. FD17 . PGP traits . Plantgrowth . Plant colonization .Maize

Introduction

The exponential growth of the world's population has led to anincreasing demand on food production, which is accompaniedby an increasing demand on fertilizers. This has not only causedan extreme rise in the price of fertilizers, but will lead to anunavoidable world shortage of, e.g., phosphate, within decades(Vance 2011). We face the same problem for water, and globalwarming makes the situation even worse. Innovative agricul-tural management systems are needed to fulfill the future globaldemand for food, feed, and fiber products while minimizingnegative atmospheric, soil, and water quality impacts (Hayatiet al. 2011; Lambin and Meyfroidt 2011).

The use of biological inoculants based on plant growth-promoting bacteria (PGPB) appears to be a promising alterna-tive to chemicals. Consequently, over the years, the utilizationof PGPB, usually either rhizosphere bacteria or endophytes, asbio-fertilizers and/or bio-pesticides has received increasing at-tention. These microorganisms may not only ensure the avail-ability of essential nutrients to plants but also enhance nutrientuse efficiency (Khalid et al. 2009). Endophytic bacteria may infuture be even more important than rhizosphere bacteria inpromoting plant growth promotion and nutrition, because theyescape competition with rhizosphere microorganisms andachieve a more intimate contact with plant tissues.

M. Naveed (*) :B. Mitter :M. Pastar :A. SessitschBioresources Unit, AIT Austrian Institute of Technology GmbH,Konrad-Lorenz-Strasse 24, 3430 Tulln, Austriae-mail: muhammad.naveed.fl@ait.ac.at

S. YousafDepartment of Environmental Sciences, Faculty of BiologicalSciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan

M. AfzalNational Institute for Biotechnology and Genetic Engineering(NIBGE), 38040 Faisalabad, Pakistan

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Proposedmechanisms bywhich PGPBmay stimulate plantgrowth and nutrition include biological nitrogen fixation(BNF), synthesis of phytohormones, environmental stressrelief, synergism with other plant–microbe interactions, inhi-bition of plant ethylene synthesis, as well as increasing avail-ability of nutrients like phosphorus, iron and other micro-elements, and growth enhancement by volatile compounds(Hardoim et al. 2008; Compant et al. 2010; Mitter et al.2013). However, the expression of such bacterial activitiesunder laboratory conditions does not necessarily guarantee thesame beneficial effects in association with plants grown undernatural conditions (Fuentes-Ramirez and Caballero-Mellado2005; Smyth et al. 2011).

The identification and selection of effective PGPB, which areeffective when applied onto plants grown in natural soils, can betime consuming, laborious and costly. The selection procedureusually involves the collection of plant-associated bacteria andsubsequent screening under various environmental conditions.Usually screenings are performed in a bottom-up approach,starting with multiple laboratory assays and tests performedunder gnotobiotic conditions. Selected strains are then tested inthe greenhouse followed by field evaluation (Khalid et al. 2004;Hynes et al. 2008). In many studies bacteria have been isolatedand screened for few plant growth-promoting capacities in thelaboratory, typically for IAA, ACC deaminase, P solubilizationor antibiotic production (Penrose and Glick 2003; Khalid et al.2004; Hynes et al. 2008; Bashan et al. 2013). However, many ofthese strains then failed to be successful when applied ontoplants grown in non-sterile soil. Strains may either fail to suc-cessfully colonize or beneficial activities may not be expressedin associationwith plants grown under greenhouse or under fieldconditions (Smyth et al. 2011).

The aim of the present study was to select a bacterial strain,which has the potential to enhance plant performance in thefield and which potentially works with different plant geno-types. We therefore tested five bacterial strains, which werepreviously isolated as root endophytes from maize (Prischlet al. 2012) and subjected them to a rigorous testing in thelaboratory addressing a multitude of characteristics, gnotobi-otic plant tests with five maize varieties as well as plantcolonization characteristics. Enterobacter sp. strain FD17was selected as most promising strain and further tested withtwomaize cultivars grown in a net house, in which plants wereexposed to natural, climatic conditions.

Materials and methods

Endophyte strains used in this study

Previously bacterial endophytes were isolated from the maizeroots by Prischl et al. (2012). Based on IAA production (qual-itative test) and ACC deaminase activity, five isolates —

Caulobacter sp. FA 13, Pantoea sp. FF 34, Sphinogobiumsp. FC 42, Pseudomonas sp. FB 12, and Enterobacter sp.FD17, were characterized in this study and tested in detail inregard to plant growth promotion and colonization. To confirmthe genus assigned based on 16S rRNA gene analysis (Prischlet al. 2012), strain Enterobacter sp. FD17 was further charac-terized by partial sequence analysis of the gyrB gene(Yamamoto and Harayama 1995; Brady et al. 2008).Sequencing was performed making use of the Sanger sequenc-ing service of the company AGOWA (Berlin, Germany).Retrieved sequences were visualized with sequence alignmenteditor package of BioEdit and identified by BLAST analysis.

Nucleotide sequence accession number

The sequence determined in this study was deposited in theGenBank database with the accession number KF147850.

Functional characterization of the endophytic strains

Phenotypic and physiological characterization

Bacterial strains from overnight grown cultures in TSA brothwere streaked on TSA agar plates and incubated at 30 °C.After 24 h, the colour and shape of colonies were noted. Cellmotility and shape of single colony was observed under lightmicroscope (Nikon, Japan).

The pH limits for bacterial growth was determined adjustedto pH values between 5 and 12 in triplicates. The dependenceof bacterial growth on different salt concentrations was deter-mined in the same medium containing 1–6 % NaCl.Furthermore, the ability to growth in methanol/ethanol as soleC source was analyzed.

Bacterial capacity to aggregate formation may positivelyaffect their dispersal and survival in the plant environment andadsorption to plant roots. The extent of aggregation formationwas measured in six replicates following the method of Madiand Henis (1989) with some modifications. Aliquots of liquidculture containing aggregates were transferred to glass tubesand allowed to stand for 30 min. Aggregates settled down tothe bottom of each tubes, and the suspension was mostlycomposed free of cells. The turbidity of each suspension wasmeasured at 540 nm (ODs) with a microplate reader (Synergy5; BioTek Instrument Inc., Winooski, USA). Cultures werethen dispersed with a tissue homogenizer for 1 min and thetotal turbidity (OD) was measured. The percentage of aggre-gation was estimated as follows:

%aggregation ¼ ODt−ODsð Þ � 100=ODt

Motility assays (swimming, swarming and twitching) wereperformed following the method of Rashid and Kornberg

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(2000). Swim plates (LB media contained 0.3 % agarose)were inoculated in triplicates with bacteria from an overnightculture on TSA agar plates grown at 30 °C with a steriletoothpick. For swarming, plates (NB media contained 0.5 %agar and glucose) were inoculated with a sterile toothpick.Twitch plates (LB broth containing 1 % Difco granular agar)were stab inoculated with a sharp toothpick to the bottom ofPetri dish from an overnight grown culture in TSA agar plates.

Biofilm formation was analyzed using overnight grownbacterial culture in 96 well microtiter plates by staining with1 % crystal violet (CV) for 45 min. To quantify the amount ofbiofilm, CV was destained with 200 μl of 100 % ethanol. Theabsorbance of 150 μl of the destained CV, which was trans-ferred into a new microtiter plate was measured at 595 nm(modified from Djordjevic et al. 2002).

Biochemical characterization

Biochemical tests such as oxidase, catalase, gelatin hydrolysisand casein hydrolysis of the selected strains were performed.Oxidase and catalase activities were tested with 1 % (w/v)tetramethyl-p -phenylene diamine and 3 % (v/v) hydrogenperoxide solution, respectively. Gelatin and casein hydrolysiswas performed by streaking bacterial strains onto a TSA platesfrom the stock culture. After incubation, trichloroacetic acid(TCA) was applied to the plates and made observation imme-diately for a period of at least 4 min (Medina and Baresi 2007).

Quantification of auxin production

Auxin production by bacterial isolates both in the presence andabsence of L-tryptophan (L-TRP) was determined colorimetri-cally and expressed as IAA equivalent (Sarwar et al. 1992).Two-day-old bacterial cells grown (28 °C at 180 rpm) in TSAbroth supplemented with 1 %L-TRP solution were harvestedby centrifugation (10,000×g for 10min). Threemilliliters of thesupernatants were mixed with 2 ml Salkowski's reagent(12 g l−1 FeCl3 in 429 ml l−1 H2SO4). The mixture wasincubated at room temperature for 30 min for colour develop-ment and absorbance at 535 nm was measured using spectro-photometer. Auxin concentration produced by bacterial isolateswas determined using standard curves for IAA prepared fromserial dilutions of 10–100 μg ml−1.

Assays for phosphorus solubilization and siderophoreproduction

Bacterial strains were evaluated for their ability to solubilizephosphates (organic/inorganic P). Aliquots (10 μl) of over-night bacterial growth culture in TSA medium were spotinoculated onto NBRI-PBP (Mehta and Nautiyal 2001) andcalcium/sodium phytate agar medium (Rosado et al. 1998).Solubilization of organic/inorganic phosphates was detected

by the formation of a clear zone around the bacterial growthspot. Phosphate solubilization activity was also determined bydevelopment of clear zone around bacterial growth onPikovskaya agar medium (Pikovskaya 1948). Bacterial iso-lates were assayed for siderophores production on the Chromeazurol S (CAS) agar medium described by Schwyn andNeilands (1987) as positive for siderophore production.

Assays for exopolysaccharide, NH3 and HCN production

For exopolysaccharide (EPS) activity (qualitative), strainswere grown on Weaver mineral media enriched with glucoseand production of EPS was assessed visually (modified fromWeaver et al. 1975). The EPS production was monitored asfloc formation (fluffy material) on the plates after 48 h ofincubation at 28±2 °C. Strains were tested for the productionof ammonia (NH3) in peptone water as described byCappuccino and Sherman (1992). The bacterial isolates werescreened for the production of hydrogen cyanide (HCN) byinoculating King's B agar plates amended with 4.4 g l−1 gly-cine (Lorck 1948). Filter paper (Whatman no. 1) saturatedwith picrate solution (2 % Na2CO3 in 0.5 % picric acid) wasplaced in the lid of a petri plate inoculated with bacterialisolates. The plates were incubated at 28±2 °C for 5 days.HCN production was assessed by the colour change of yellowfilter paper to reddish brown.

Assays for poly-hydroxybutyrate (PHB)and n -acyl-homoserine lactone (AHL) production

The bacterial isolates were tested for PHB production(qualitative) following the viable colony stainingmethods usingNile red and Sudan black B (Juan et al. 1998; Spiekermannet al. 1999). The LB plates with overnight bacterial growthwere flooded with 0.02 % Sudan black B for 30 min and thenwashed with ethanol (96 %) to remove excess strains from thecolonies. The dark blue coloured colonies were taken as posi-tive for PHB production. Similarly, LB plates amended withNile red (0.5 μl ml−1) were exposed to UV light (312 nm) afterappropriate bacterial growth to detect PHB production.Colonies of PHA-accumulating strains showed fluoresce underultraviolet light. The bacterial strains were tested for AHLproduction following the method modified from Cha et al.(1998). The LB plates containing 40μgml−1 X-Gal were platedwith reporter strains (Agrobacterium tumefaciensNTL4.pZLR4). The LB plates were spot inoculated with10 μl bacterial culture and incubated at 28±2 °C for 24 h.Production of AHL activity is indicated by a diffuse blue zonesurrounding the test spot of culture. A. tumefaciens NTL1(pTiC58ΔaccR) was used as positive control and plate withoutreporter strain was considered as negative control.

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Enzyme hydrolyzing activities

Bacterial hydrolyzing activities due to amylase, cellulase,chitinase, lipase, pectinase, protease and xylanase werescreened on diagnostic plates after incubation at 28 °C.Amylase activity was determined on agar plates followingthe protocol Männistö and Häggblom (2006). Formation ofan opaque halo around colonies indicated lipase activity.Cellulase and xylanase activities were assayed on plates con-taining (per liter) 5 g of carboxymethyl cellulose or birchwood xylan, 1 g of peptone and 1 g of yeast extract. After10 days of incubation, the plates were flooded with gram'siodine staining and washing with 1 M NaCl to visualize thehalo zone around the bacterial growth (modified from Teatherand Wood 1982). Chitinase activity of the isolates was deter-mined as zones of clearing around colonies following themethod of Chernin et al. (1998). Protease activity was deter-mined using 1 % skimmed milk agar plates, while lipaseactivity was determined on peptone agar medium. Formationof halo zone around colonies was used as indication of activity(Smibert and Krieg 1994). Pectinase activity was determinedon nutrient agar supplementedwith 5 g l−1 pectin. After 1weekof incubation, plates were flooded with 2 % hexadecyltrimethyl ammonium bromide solution for 30 min. The plateswere washed with 1 MNaCl to visualize the halo zone aroundthe bacterial growth (Mateos et al. 1992).

Antagonistic activities against plant pathogenic bacteria, fungiand oomycetes

The antagonistic activities of bacterial isolates were screenedagainst plant pathogenic bacteria (A. tumefaciens ,Pseudomonas syringae , Streptococcus pneumoniae), fungi(Fusarium caulimons , Fusarium graminarium , Fusariumoxysporum , Fusarium solani , Rhizoctonia solani ,Thielaviopsis basicola) and oomycetes (Phytophthora infestans ,Phytophthora citricola , Phytophthora cominarum ). Forantibacterial assays, the bacterial isolates and pathogen werecultivated in TSA broth at 30 °C for 24 h. The bacterialisolates were spot-inoculated (10 μl aliquots) on TSA platespre-seeded with 100 μl tested pathogen. The plates wereincubated at 28 °C for 48 h and clear zones of inhibitionwere recorded.

Antagonistic activity of the bacterial isolates against fungiand oomycetes was tasted by the dual culture technique onpotato dextrose agar (PDA) and yeast malt agar (YMA) media(Dennis and Webster 1971). A small disk (5 mm) of targetfungus/oomycetes was placed in the center of petri dishes ofboth media. Aliquots of 10 μl of overnight bacterial culturesgrown in TSAwere spotted 2 cm away from the center. Plateswere incubated for 14 days at 24 °C and zones of inhibitionwere scored.

Effect of endophytic strains on maize germination

Inoculants of the selected strains were prepared in 50 ml TSAbroth in 100-ml Erlenmeyer flasks and incubated at 28±2 °Cfor 48 h in the orbital shaking incubator (VWR International,GmbH) at 180 rev min–1. The optical density of the broth wasadjusted to 0.5 measured at 600 nm using spectrophotometer(Gene Quant Pro, Gemini BV, The Netherlands) to obtain auniform population of bacteria (108–109 colony-forming units(CFU) ml–1) in the broth at the time of inoculation.

Maize seeds were surface-sterilized with 70 % ethanol(3 min), treated with 5 % NaHClO for 5 min, and followedby washing three times with sterile distilled water (1 min eachtime). The efficacy of surface sterilization was checked byplating seed, and aliquots of the final rinse onto LB plates.Samples were considered to be successfully sterilized whenno colonies were observed on the LB plates after inoculationfor 3 days at 28 °C. Surface-disinfected seeds of differentmaize cultivars (Helmi, Morignon, Pelicon, Peso and Cesor)were immersed in the bacterial suspensions for 30 min. Thebacterized seeds were deposited onto soft water-agar plates(0.8 %, w/v agar) and plates were placed in the dark at roomtemperature (24±2 °C). After 96 h the percentage of germi-nated seeds was scored. Surface-sterilized seeds, but not bac-terized (treated in TSA broth), served as the germinationcontrol.

In vitro screening of efficient strains under axenic conditions

A growth chamber experiment was conducted on maize toscreen the selected strains for their growth promoting activityunder gnotobiotic conditions. We used specially designed glasstubes with beaded rim (Duran group, DURAN GmbH, Mainz,Germany) for the experiment. The glass tubes were covered withlid to generate fully axenic conditions (no exposure to anyenvironmental factors). Bacterial inoculant production and seedtreatment were done as described above. As control, seeds weretreatedwith sterilized TSA broth. Treated seedswere placed ontowater-agar plates for germination. After 5 days, germinatedseedlings (3–5 cm long) were transferred in the sterilized glasstubes containing sterilized 20 ml MS (Murashige and Skoog)medium (Duchefa Biochemie, The Netherlands) (4.8 g l−1) andplaced at 25±2 °C set at a 16-h light and 8-h dark period, with alight intensity of 350 μmol m−2 s−1. Data regarding shoot/rootlength and biomass were recorded after 24 days. Colonization ofinoculant strains was scored by re-isolation of endophytes. Onegram of plant shoot was homogenized with a pestle and mortarin 4 ml of 0.9 % (w/v) NaCl solution. The number of cultivableendophytes in maize shoot, expressed in CFU per gram (freshweight), was determined by spreading serial dilution up to 10−4

(0.1 ml) of homogenized surface-sterilized plant material ontoTSA (DIFCO Laboratories, Detroit, MI, USA) agar medium.Four replicates for each treatment were spread on the agar plates

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and incubated for 5 days at 28 °C. Twenty colonies per treatmentwere randomly selected and their identity with the inoculantstrain was confirmed by restriction fragment length polymor-phism (RFLP) analysis of the 16S–23S rRNA intergenic spacer(IGS) region (Reiter et al. 2002).

Net house experiment

On the basis of the results from tests performed under axenicconditions, strain FD17 was selected for further evaluation ina pot trial, in which plants were grown in large containersexposed to natural environmental conditions.

Maize plants were grown in soil collected from agricultural(maize) fields in Fischamend, Lower Austria, Austria. The soilwas silty clay loam and had the following characteristics: 12 %sand, 61 % silt, 27 % clay, pH 6.5, 3.3 % total carbon, 0.18 %total nitrogen, 0.13 mg g−1 available phosphorus, 0.066 mg g−1

extractable potassium.Surface-disinfected seeds of two maize cultivars (Morignon

and Peso) were immersed in bacterial suspension (prepared asdescribed above) for 1 h. For the un-inoculated control, seedswere treated with sterilized TSA broth. Seeds were sown in aplastic tray (wiped with ethanol) and 12-day-old seedlings weretransferred into containers filled with 45 kg soil (two plants ineach container) and placed in a net house and exposed to naturalenvironmental conditions.

Weather conditions, i.e., precipitation, temperature andrelative humidity, were recorded by 'Zentralanstalt fürMeteorologie und Geodynamik' (ZAMG) during the cropgrowth period and described in Fig. 1. There were threereplicated and the pots were arranged in a completely

randomized design. Recommended dose of NPK fertilizers(160–100–60 kg ha−1) were applied in each container and tapwater was applied to the container for irrigation wheneverneeded.

Data of photochemical efficiency of PSII was recorded atflowering stage using handy PEA (Hansatech InstrumentsLtd., England) in the mid of July where daytime temperaturevaried from 30 °C to 35 °C. The PSII efficiency in terms ofFv/Fm was calculated from the data. Growth and yield con-tributing parameters were recorded at maturity. The plantswere harvested 140 days after planting.

Rhizosphere and endophytic colonization of roots, stems andleaves by the inoculant strain were determined by plate countingusing TSA plates. Root, stem and leave samples were washed,surface sterilized (as described above) and used for inoculantstrain recovery (colonization). For this, samples were crushed in0.9 % (w/v) NaCl solution, shaken with a pulsifier (MicrogenBioproducts Ltd., UK) for 30 s, and different dilutions werespread on TSA plates. Bacterial colonies were counted after4 days of incubation at 28±2 °C. The selected colonies wereidentified and confirmed by IGS region-based RFLP analysis.

Statistical analyses

The data of plant growth parameters and colonization weresubjected to analyses of variance. The means were comparedby least significant difference (LSD) test (p <0.05) to detectstatistical significance among treatment (Steel et al. 1997). Allof the statistical analyses were conducted using SPSS softwareversion 19 (IBM SPSS Statistics 19, USA).

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Precipitation (mm) Temp °C Relative HumidityFig. 1 Weather conditions dataduring the crop growth periodobtained from ZAMG, Austria

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Results

Gyrase B sequence analysis of strain FD17

The partial nucleotide sequences of gyrB gene of strain FD17revealed 99 % identities with the Genbank entry CP002886.1,Enterobacter cloacae (987 bp).

Functional characterization of endophyte isolates basedon laboratory assays

A range of features known to contribute to plant growthpromotion, stress tolerance or biocontrol was tested. Theresults of characterization are summarized in Table 1. Allstrains showed IAA production (ranging from 1.83 to10.33 μg ml−1 IAA equivalent), NH3 and siderophore pro-duction (qualitative) but with variable degree of efficacy. Onlystrain FB12 was able to produce AHL. Three strains (FF34,FB12 and FD17) showed P solubilization and PHB produc-tion, whereas only FB12 was able to produce HCN. Likewise,strain FB12 showed positive for all biochemical charactersmentioned in the Table 1. Strain FA13 produced EPS moreefficiently compared to FC42 and FD17. All strains showedlipase activity, while none of the strain produced amylase andprotease activities. Pectinase activity was observed withstrains FF34, FB12 and FD17. All strains were positive forcellulase and xylanase activity except strain FF34. Chitinase wasproduced by strains FB12 and FD17. Strain FD17 exhibitedbetter aggregate stability, chemotaxis and biofilm formation ascompared to other strains. Strains FB12 and FD17 showedantagonistic activity against various bacterial pathogens as com-pared to other strains. All strains showed antagonism againstdifferent fungal pathogens and oomycetes but with variabledegree of efficacy. Strain FD17 showed highest antagonismagainst F. caulimons , F. solani and P. citricola.

Effect on maize germination

Inoculation of maize seeds with endophytic bacteria increasedthe germination rate of all cultivars by 20–40 % compared tothe un-inoculated control (Fig. 2). Maximum increase wasobserved by inoculation with strain FD17 (40 %) in maizecv. Morignon followed by strains FF34, FA13, FB12 andFC42. The minimum increase was recorded by inoculationwith strain FB12 and strain FC42 in the maize cultivars Helmiand Cesor, respectively.

In vitro screening of efficient strains under axenic conditions

All strains significantly increased the seedling growth com-pared to the control (Table 2). For three cultivars (Peso,Morignon, Cesor), the strain FD17 performed best in regardto root and shoot length production (Table 2), while for Helmi

and Pelicon higher values were recorded with other strains.The maximum root length formation was found with cultivarPeso and strain FD17 (27.8 cm; control 19.25 cm). StrainFB12 inhibited root formation of cultivar Helmi, but promotedroot formation of other cultivars. The maximum shoot lengthformation was observed with strain FD17 and cultivarMorignon (34.75 cm; control 27.75 cm). Again, some strainsdid not promote shoot formation of some cultivars, but had apositive effect on others (Table 2). All strains significantlypromoted biomass production (Table 3). Strain FD17 wasbeneficial for all cultivars, whereas other strains showed morecultivar-specific responses. The highest shoot biomass wasfound with Morignon and strain FD17 (increased by 96 %compared to control treatment). Generally, cultivar Helmishowed low response to bacterial inoculation (Table 3).

Figure 3 shows that bacterial strains efficiently colonized allmaize cultivars; viable cell numbers ranged from 1.20×104 to2.93×107 cells g−1 fresh plant material. Maximum colonizationwas recorded by inoculation with strain FD17 in the shootinterior of Morignon followed by Peso. Strain FD17 was ableto colonize all cultivars quite efficiently, whereas other strainsshowed more variable colonization characteristics. Peso wasthe only cultivar well colonized by all strains (Fig. 3).

Net house experiment

Based on the results obtained under axenic conditions, strainFD17 was selected for further evaluation for its performanceunder conditions close to those encountered in the field. Thenet house is a wire construct without glass roof, but is coveredby a net only, which is permeable for rain. Plants encounterednatural climate conditions. Data on climate conditions (Fig. 1)revealed precipitation ranges of 0–42.9 mm, temperatureranges of 4.6–34.6 °C and relative humidity values of 50.1–95.9 during the crop growth period.

Inoculation with strain FD17 led to a significant increase inleaf area of both cultivars (20 % and 13 %, respectively;Table 4). Similarly, biomass (leaf dry weight) was increasedby 27 % and 23 % in the cultivars Peso and Morignon,respectively, as compared to the control. Similarly, root andplant dry biomass and plant height were significantly en-hanced as well as cob weight (35 % and 42 % increase inPeso and Morignon, respectively, as compared to control).

Strain FD17 affected plant physiological characteristicssuch as increase of chlorophyll fluorescence (maximum in-crease of 9 % in cultivar Peso) and reducing the time neededfor the onset of flowering (up to 10 days in cultivar Peso)(Table 4).

Strain FD17 efficiently colonized rhizosphere, root, shootand leaf interior of both maize cultivars and no significantdifferences in the viable cell number were encountered.Higher colonization was found in the rhizosphere and rootinterior as compared to the shoot and leaf interior (Table 5).

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Table 1 Physico-chemical and growth-promoting characteristics of maize endophytes

Characteristics Caulobactersp. (FA13)

Pantoeasp. (FF34)

Sphinogobiumsp. (FC42)

Pseudomonassp. (FB12)

Enterobactersp. (FD17)

Phenotypic and physiological characterization

Colony colour Gray Yellow Yellow Gray Creamy white

Colony morphology Round Round Round Round Round

Bacterial growth conditionsa

NaCl

2 % + + + + +

6 % − + − − +

pH

5 + + + + +

12 + − − + +

Motility/chemotaxisb

Swimming + + − ++ +++

Swarming − − − − +

Twitching + + − + +

Biofilm formation

OD (600 nm) 0.92±0.04 0.59±0.02 0.95±0.08 0.57±0.08 0.95±0.04

Biofilm (595 nm) 0.23±0.02 0.22±0.03 0.08±0.01 0.08±0.04 0.83±0.06

Aggregate stability (%) 35.91±2.57 26.07±0.88 32.61±2.13 36.38±1.48 40.22±1.99

Biochemical characterizationa

Catalase + + + + +

Oxidase − − − + −Casein − − − + −Gelatin − + − + +

Methanol + − − + −Ethanol + − − + −

Growth promoting characterizationb

ACC deaminase + + + ++ +++

Auxin production (IAA equivalent, μg ml−1)

Without L-TRP 1.74±0.18 10.33±0.35 4.89±0.78 1.63±0.65 7.54±1.02

With L-TRP 16.13±1.05 95.34±2.14 38.41±1.78 7.26±1.05 12.30±0.98

P solubilization (inorganic/organic P)

Ca3(PO4)2 − ++ − + +++

CaHPO4 − ++ − + +++

Ca(H2PO4)2 − ++ − ++ +++

Ca phytate − ++ − ++ +++

Na phytate − ++ − ++ +++

Exopolysaccharide ++ − + − +

HCN production − − − + −NH3 production + + + + +

Siderophore production +++ + + ++ +++

AHL − − − + −PHB − + − + +

Enzyme hydrolyzing activityb

Amylase − − − − −Cellulase + − + + ++

Chitinase − − − + +

Lipase ++ + + +++ ++

Pectinase − + − + +

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Table 1 (continued)

Characteristics Caulobactersp. (FA13)

Pantoeasp. (FF34)

Sphinogobiumsp. (FC42)

Pseudomonassp. (FB12)

Enterobactersp. (FD17)

Protease − − − − −Xylanase + − +++ + ++

Antagonistic activities against plant pathogenic bacteria, fungi and oomycetesb

Anti-bacterial activity

A. tumefaciens − − − ++ +

P. syringae − − − +++ +

S. aureus − − − + −Anti-fungal activity

F. caulimons ++ + + ++ +++

F. graminarium + + + + ++

F. oxysporum + ++ + ++ ++

F. solani ++ + ++ ++ +++

R. solani + + + ++ ++

T. basicola + + + ++ +

Anti-oomycete activity

P. infestans + + + ++ ++

P. citricola + + + ++ +++

P. cominarum + + + ++ ++

Results are obtained from 4 to 6 replicatesa−, absent; +, presentb +, low efficiency; ++, medium efficiency; +++, high efficiency

0

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Control Caulobacter sp.FA13

Pantoea sp.FF34

Sphingobium sp.FC42

Pseudomonassp. FB12

Enterobacter sp.FD17

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of

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s ge

rmin

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Helmi Peso Pelicon Morignon Cesor

Fig. 2 Effect of inoculation with selected bacterial endophyte on germination of different maize cultivars under axenic conditions. Data represents theaverage of three replicates ± SE

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Discussion

The use of PGPB is currently gaining worldwide interest as apromising alternative to the use of potentially polluting chem-ical fertilizers and pesticides, particularly in organic produc-tion. PGPB may employ different mechanisms to enhanceseed germination, root development or to improve mineralnutrition and water utilization (Dobbelaere et al. 2003; Mitteret al. 2013). Generally, a bottom-up approach is employed toselect strains, which are promising for field application.Initially, laboratory-based tests of various activities potentiallyinvolved in plant growth promotion are used, followed ingreenhouse studies and in some cases by field tests (Khalidet al. 2004; Smyth et al. 2011; Bulgarelli et al. 2013). Oftenonly few characteristics are tested in the laboratory and strainsselected in the laboratory have in many cases proven not toshow the expected success when tested on plants grown innon-sterile soil (Barret et al. 2011). This might be due to thefact that an inoculant strain needs to be able to establish in ahighly competitive environment, a characteristic, which israrely addressed in initial screenings. They also might be poorplant colonizers. Furthermore, several mechanisms involvedin plant growth promotion might be needed to confer the

beneficial effects and/or the desirable activities (observedunder laboratory conditions) might only be expressed underspecific conditions (reviewed for Azospirillum sp. by Bashanand de-Bashan 2010).

In this study, we rigorously tested several strains previouslyisolated as endophytes from maize roots (Prischl et al. 2012).Based on IAA production and ACC-deaminase capability,five bacterial strains (used in the present study) were selectedfor rigorously testing their plant growth promotion potential inlaboratory and plant assays as well as their plant colonizationcapacities (Table 1). Based on these numerous characteristics,we selected Enterobacter sp. strain FD17 and confirmed itsplant growth-promoting effects with two maize cultivarsgrown in regular (maize) field soil and subjected to naturalclimatic conditions.

The five endophyte strains showed highly variable growth-promoting traits and plant growth promotion effects whentested in laboratory trials and plant assays. All of the isolateswere able to produce IAA equivalent, but with variable effi-ciency in the presence and absence of L-TRP. Under axenicconditions, inoculation improved root–shoot length and bio-mass of different maize cultivars. It has been described that rootgrowth can be stimulated or inhibited depending on the

Table 2 Effect of inoculation with selected bacterial endophyte on root and shoot length of different maize cultivars under axenic conditions

Strain/Maize cv. Root length (cm) Shoot length (cm)

Helmi Peso Pelicon Morignon Cesor Helmi Peso Pelicon Morignon Cesor

Control 18.52 i–k 19.25 g–i 15.50 l–n 16.50 k–m 14.25 n 14.77 n 25.12 jk 24.25 kl 27.75 hi 23.25 lm

Caulobacter sp. FA13 26.37a–c 25.25 b–d 25.75 a–d 19.25 g–i 20.00 gh 26.50 ij 33.75 a–d 31.50 e–g 32.92 b–e 30.50 g

Pantoea sp. FF34 26.55 ab 22.50 ef 16.50 k–m 16.75 j–m 17.25 i–l 28.60 h 28.87 h 28.32 h 32.12 d–g 32.50 c–f

Sphinogobium sp. FC42 18.67 h–j 26.75 ab 27.50 a 17.50 i–l 15.50 l–n 22.50 m 31.15 fg 33.92 a–c 32.15 d–g 32.40 c–f

Pseudomonas sp. FB12 14.75 mn 24.00 de 24.37 c–e 21.00 fg 21.40 fg 15.25 n 32.45 c–f 33.75 a–d 32.50 c–f 32.27 c–f

Enterobacter sp. FD17a 24.67 b–d 27.85 a 22.50 ef 21.57 fg 21.45 fg 26.35 ij 34.37 ab 32.15 d–g 34.75 a 32.82 c–f

Means sharing similar letter(s) in a column for each parameter do not differ significantly at p=0.05. The data is average of four replicatesa Efficient strain selected for containment experiment

Table 3 Effect of inoculation with selected bacterial endophyte on root and shoot biomass of different maize cultivars under axenic conditions

Strain/Maize cv. Root biomass (g) Shoot biomass (g)

Helmi Peso Pelicon Morignon Cesor Helmi Peso Pelicon Morignon Cesor

Control 0.81 ij 0.86 gh 0.68 k 0.76 jk 0.57 l 0.77 p 1.16 kl 0.87 no 1.01 m 0.79 op

Caulobacter sp. FA13 1.08 g 1.28 ef 1.04 g 1.26 ef 1.06 g 1.05 m 1.63 e 1.62 e 1.74 d 1.36 hi

Pantoea sp. FF34 1.39 cd 1.56 a 0.95 h 0.95 h 0.89 gh 1.30 ij 1.77 cd 1.09 lm 1.44 gh 1.34 i

Sphinogobium sp. FC42 1.24 ef 1.24 ef 1.20 f 1.10 g 1.07 g 1.08 lm 1.69 de 1.59 ef 1.75 d 1.51 fg

Pseudomonas sp. FB12 0.91 h 1.46 bc 1.10 g 1.29 ef 1.06 g 0.91 n 1.76 d 1.32 ij 1.92 ab 1.52 fg

Enterobacter sp. FD17a 1.46 bc 1.57 a 1.31 de 1.49 ab 1.26 ef 1.24 jk 1.86 bc 1.63 e 1.98 a 1.52 fg

Means sharing similar letter(s) in a column for each parameter do not differ significantly at p=.0.05. The data is average of four replicatesa Efficient strain selected for containment experiment

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concentration of IAA produced by bacteria (Zúñiga et al. 2013).This is also in line with our results, where strain FF34 showedhighest IAA production, but induced less root biomass thanother strains producing less IAA. However, we have to beaware that we only have results of laboratory assays and haveno information on in planta activities. Glick et al. (1998)introduced a model in which IAA synthesis also plays a criticalrole in ACC synthesis, the immediate precursor of ethylene.

Therefore, high concentrations of IAA lead to high levels ofethylene, decreasing root length (Strader et al. 2010). Recently,Bhattacharjee et al. (2012) observed an increase in plant bio-mass of rice cultivars due to IAA and ACC deaminase produc-tion by PGPB upon inoculation. The role of ACC deaminase indecreasing ethylene levels by the enzymatic hydrolysis of ACCinto α-ketobutyrate and ammonia has been documented as oneof the major mechanisms of PGPR in promoting root and plant

c

bc

c cbc

ab

a

bc

a

a

c c c c

ab

bc

b

b

bc

a

bc

b

cbc bc

4.E+02

3.E+06

6.E+06

9.E+06

1.E+07

2.E+07

2.E+07

2.E+07

2.E+07

3.E+07

3.E+07

Caulobacter sp.FA13

Pantoea sp. FF34 Sphingobium sp.FC42

Pseudomonas sp.FB12

Enterobacter sp.FD17

CF

U/g

sho

ot f

resh

bio

mas

s

Helmi Peso Pelicon Morignon Cesor

Fig. 3 Persistence of selected endophytic strains in the shoot interior of different maize cultivars under axenic conditions. Bars sharing similar letters donot differ significantly at p =0.05

Table 4 Effect of inoculation with endophytic strain FD17 on physiology, growth parameters and yield of two maize cultivars grown in pots in field soiland exposed to natural climatic conditions (net house experiment)

Parameters/treatment Peso Morignon

Un-inoculated Inoculated with FD17a Un-inoculated Inoculated with FD17a

Fv/Fm 0.69 c 0.75 b (8.69) 0.73 bc 0.79 a (8.22)

Time to onset of flowering (days) 65.33 b 55.00 a (15.81) 70.67 d 66.33 bc (6.14)

Plant height (cm) 192.33 d 208.00 ab (8.15) 196.69 cd 213.68 a (8.64)

No. of leaves plant−1 12.33 c 14.00 ab (13.54) 13.17 bc 14.67 a (11.39)

Leaf area (cm2) 494.26 d 556.27 bc (12.55) 512.39 cd 617.11 a (20.44)

Leaf dry weight (g) 22.21 c 28.16 b (26.79) 28.09 b 34.56 a (23.03)

Plant dry biomass (g) 114.18 c 153.77 b (34.67) 160.46 b 223.14 a (39.06)

Root dry biomass (g) 17.26 c 24.34 b (41.02) 19.73 c 28.28 a (43.34)

Cob weight (g) 115.28 c 155.83 b (35.18) 123.71 c 176.23 a (42.45)

Means sharing similar letter(s) in a row for each parameter do not differ significantly at p =0.05. The data are based on three replicates. Values in bracketsare % increase over controlaEnterobacter sp. strain FD17

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growth (Glick et al. 1998). In our study, all strains producedACC deaminase, which was most active in Enterobacter sp.strain FD17 and might be needed to confer beneficial effects.

Phosphorus is very important for normal plant growth anddevelopment. The inoculationwith themicroorganisms that havethe ability to solubilize P could enhance the quantity of effectiveP and increase crop yields (Yao 2004). The strains used in thisstudy have the ability to solubilize tricalcium phosphate (TCP)and dicalcium phosphate and, therefore, could be effective forimproving crop yield under natural conditions. This can beattributed to the release of different organic compounds in therhizosphere by microorganisms may be important in the solubi-lization of various inorganic P compounds (Scervino et al. 2010).However, contrary to this, Collavino et al. (2010) reported thatin vitro P solubilization by microorganisms was not necessarilyassociated to the promotion of plant growth. Very recently,Bashan et al. (2013) suggested that TCP, is relatively weak andunreliable as a universal selection factor for isolating and testingphosphate-solubilizing bacteria (PSB) for enhancing plantgrowth. Out of five strains, three were positive for EPS andPHB production. Again, strain FD17 showed the highest activ-ities. Bacterial EPS and PHB have been shown to provideprotection from such environmental insults as desiccation, pre-dation, and the effects of antibiotics (Gasser et al. 2009; Staudtet al. 2012). They also contribute to bacterial aggregation, sur-face attachment, and plant–microbe symbiosis (Laus et al. 2005).

Bacterial survival and colonization in the plant environmentare necessary for plant growth and yield. Recently, Zúñiga et al.(2013) described that the cell-to-cell communication (QS) sys-temmediated by AHL is implicated in rhizosphere competenceand colonization of Arabidopsis thaliana by B. phytofirmansPsJN. Chemotaxis (Motility), aggregate stability, and biofilmformation are important traits for root surface colonization(Danhorn and Fuqua 2007). It has been suggested that a pre-requisite for the effective colonization of roots is positivechemotaxis towards root exudates (Bhattacharjee et al. 2012).A positive chemotaxis of selected strains therefore suggests thatmaize seed and root exudates release compounds that attractbacteria towards the plant leading to colonization. In the presentstudy, only FB12 showed AHL-based QS signaling, however,other communications might be involved. Aggregation andbiofilm formation were common traits in all tested strains. Inthe case of motility, four strains were positive for swimmingwhile FD17 only showed swarming.

Volatile compounds such as ammonia and HCN produced bya number of rhizobacteria were reported to play an importantrole in biocontrol (Brimecombe et al. 2001). Production ofammonia was commonly detected in all selected isolates; how-ever, only Pseudomonas sp. strain FB12 was able to produceHCN. Another important trait of PGPB — that may indirectlyinfluence the plant growth— is the production of siderophores.They bind to the available form of iron Fe3+ in the rhizosphere,thus making it unavailable to the phytopathogens and protectingthe plant health (Ahmad et al. 2008). Siderophores are known formobilizing Fe and making it available to the plant. In the presentinvestigation, all isolates showed multiple PGP activities includ-ing siderophore production and antifungal activities against oneor more test fungi. Additionally, strains FD17 and FB12 pro-duced chitinase and various other hydrolytic enzymes, whichmight help bacterial cells for plant colonization as well as forantifungal activities. Furthermore, during plant invasion, bacte-rial cells may produce hydrolytic enzymes that are recognized bythe plant, triggering the plant's resistance system (Trotel-Azizet al. 2008).

Based on our rigorous testing, taking various potential plantgrowth-promotingmechanisms into account as well as differentplant genetic backgrounds and colonization ability, we selectedstrain FD17, which also performed well under conditions re-sembling the situation in the field. Soil is a complex system andvarious biotic and abiotic factors may influence the behavior ofparticular strains in this environment. Further, it is well knownthat various stress factors frequently impact the plant and thusalter the allocation of photosynthates in the rhizosphere thatmay lead to changes in below-ground microbial communitiesand their interaction with the plant (Compant et al. 2010).Flavonoids are found within the plants that constitute a largepart of root exudates (Cesco et al. 2012). Plant root colonizationby the bacteria is considered as a primary step towards thesuccessful initiation of the plant–microbe interaction (Baiset al. 2006). Colonization in turn depends on bacterial motilitytowards the plant root release or exudates. Root exudates are arich source of nutrients for PGPB that undergo chemotaxis andcolonize the plant root surface (Bhattacharjee et al. 2012).Strain FD17 was able to successfully compete with the naturalmicroflora and successfully colonized the plant environment inaddition to promoting plant growth.

Endophytic bacteria have been isolated from roots, leaves,stems and fruits by a number of researchers. In the present

Table 5 Colonization of strain FD17 in rhizosphere, root, stem and leaves of two maize cultivars (net house experiment)

Maize cv./Plantcompartment

Rhizosphere(cfu g−1 dry wt)

Root interior(cfu g−1 dry wt)

Shoot interior(cfu g−1 dry wt)

Leaf interior(cfu g−1 dry wt)

Peso 4.07×104 a 3.39×104 a 1.63×103 b 1.16×102 c

Morignon 9.85×104 a 8.59×104 a 3.72×103 b 6.23×102 bc

Means sharing similar letter(s) in a column/row do not differ significantly at p =0.05. The data are based on three replicates

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study, endophytic bacteria have been isolated from the maizeroots. However, limited knowledge is available on plant seedas a source of endophytic bacteria, and their beneficial effectson plant growth rarely been described under natural condi-tions. Bacterial endophytes can be transferred from the plantenvironment to the seeds and may act as natural biofertilizersto the new plantlets early from seed germination (Puente et al.2009; Ruiza et al. 2011).

Microbial inoculants on the basis of plant growth promotingor biocontrol agents have a great potential for sustainable cropproduction and ecofriendly environment management (Berg2009). In our investigation, the endophytic strain FD17 in-creased growth and yield parameters of both maize cultivarsunder natural conditions.Maize, one of themost important cropspecies (C4), is known to be susceptible to moderate droughtand relatively high temperature, and C4 photosynthetic effi-ciency declines with temperature at or above 35 °C (Maiti1996). In the present study, photosynthesis (Fv/Fm) was mea-sured in the middle of July when temperature reached up to35 °C and observed significant differences between the inocu-lated treatment and the control. It is likely that endophyticbacteria while living inside plant tissues evoked various phys-iological processes to help plants to sustain photosynthesis andplant growth. Another remarkable fact was the ability of strainFD17 to influence the time needed for flowering again indicat-ing the interaction between plant and microbe in regard to plantphysiology.

The agricultural resources that commonly limit crop yieldincreases include water and nutrients, especially nitrogen (N)and phosphorous (P) (Sinclair and Rufty 2012). The interac-tions between AM fungi and bacteria in soil are of significantimportance. Perhaps fortuitously, the mycorrhizal fungi that areknown to form associations with more than 80 % of plantspecies, often enhance nutrient and water uptake. PGPR areable to increase AM fungal development by affecting rootcolonization as well as by enhancing plant N and P uptake(Richardson et al. 2009). Production of EPS by PGPB signif-icantly enhanced the attachment of bacteria tomycorrhizal rootsand AM fungal structures that influence the movement ofbacteria in the rhizosphere (Bianciotto et al. 2001). Soil mi-crobes are able to produce products that enhance the amounts ofroot exudates resulting in the activation of AM hyphae andhence higher rate of root colonization (Barea et al. 2005).However, further research is needed on the more detailedillustration of interactions between the host plant, AM fungiand soil bacteria by using different molecular techniques toenhance ecosystem productivity.

In this study, all the cultivars tested responded differently toinoculation with different endophyte isolates. Interestingly, onecultivar, Peso, was highly colonized by all strains, but plantgrowth promotion was only to a limited extent correlated withhigh colonization. However, strain FD17 was very efficient incolonizing different varieties and was also the most efficient

plant growth promoter. Efficient colonization of cv. Morginonby strain FD17 indicates the specific cultivar colonizing capacityof the bacteria. Variety-specific effects on bacterial colonizationare only poorly understood, and the genetic basis for these effectsneeds to be elucidated. Different crop species and varieties mightproduce different types of root exudates, which could triggerspecific strains or taxa (Bais et al. 2006; Andreote et al. 2010).Pillay and Nowak (1997) and Montañez et al. (2012) alsoreported that bacterization benefits depend on plant species, thecultivar and growth conditions. Moreover, in the present study,the TSAwas not entirely removed from the bacterial suspensionas previously described by Pillay and Nowak (1997). It mightinfluence the indigenous bacterial community and, thus indirect-ly had effects on plants.

In conclusion, Enterobacter sp. strain FD17 has high poten-tial to confer beneficial effects to field-grown maize, and it canbe assumed that plant growth promotion is due to a combina-tion of IAA, ACC deaminase and siderophore production andother nutrient providing activities. However, multi-sites fieldtrials with strain FD17 need to be performed combined with thedevelopment of an appropriate formulation and applicationtechnology to warrant successful performance in the field.

Acknowledgements The authors gratefully acknowledge the HigherEducation Commission (HEC) of Pakistan for financial support. Wethank Anton Grahsl for help with the net house experiment and Dr.Günter Brader for providing a reporter strain (A. tumefaciens NTL4.pZLR4) and positive control (A. tumefaciens NTL1. pTiC58ΔaccR) forthe AHL assay. The authors also thank Prof. Holger Bohlmann, Divisionof Plant Protection, University of Natural Resources and Applied Sci-ences, Vienna for providing fungal pathogen strains.

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