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Biol Fertil Soils (2014) 50:983–990 DOI 10.1007/s00374-014-0920-0

ORIGINAL PAPER

Endophytic bacteria from selenium-supplemented wheat plants could be useful for plant-growth promotion, biofortification and Gaeumannomyces graminis biocontrol in wheat production Paola Durán & Jacquelinne J. Acuña & Milko A. Jorquera & Rosario Azcón & Cecilia Paredes & Zed Rengel & María de la Luz Mora

Received: 23 December 2013 / Revised: 8 April 2014 / Accepted: 18 April 2014 / Published online: 6 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract In this study, we isolated putative plant-growthpromoting endophytic bacteria from selenium-supplemented wheat grown under field conditions. These bacterial strains belonged to Bacillus, Paenibacillus, Klebsiella, and Acinetobacter genera and showed genetic similarly with rhizospheric bacteria isolated in the same Andisol soil and with other endophytic strains previously reported. Strains isolated from selenium-supplemented wheat were highly tolerant to elevated selenium concentration (ranged from 60 to 180 mM), and showed potential plant-growth-promoting capabilities (auxin and siderophore production, phytate mineralization, and tricalcium phosphate solubilization). In addition, some strains like Acinetobacter sp. (strain E6.2), Bacillus sp. (strain E8.1), Bacillus sp., and Klebsiella sp. (strains E5 and E1) inhibited the growth of Gaeumannomyces graminis mycelia in vitro at 100, 50, and 30 %, respectively. These endophytic microorganisms would be useful for dual purposes: selenium biofortification of wheat plants and control of G. graminis, the principal soil-borne pathogen in volcanic soils from southern Chile. P. Durán : J. J. Acuña : M. A. Jorquera : C. Paredes : M. de la Luz Mora (*) Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Avenida Francisco Salazar, 01145 Temuco, Araucanía, Chile e-mail: [email protected] R. Azcón Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín (CSIC), Profesor Albareda n° 1, 1808 Granada, Spain Z. Rengel Soil Science and Plant Nutrition, School of Earth and Environment, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

Keywords Biofortification . Selenium . Endophytes . Gaeumannomyces graminis . PGPR

Introduction Crop production in southern Chile is based on ash-derived volcanic soils (Andisols) that have low selenium (Se) availability, producing plants with low Se content. Currently, Se is being studied as an essential micronutrient because of antioxidant capacity and beneficial effects on human health. Selenium dietary deficiency increases the incidence of important human diseases such as cancer and HIV (Bordoni et al. 2008; Meplan and Hesketh 2012). Hence, our research group has focused its efforts on the study of Se biofortification in crops and pastures. Coating seeds with Se increased both Se content and the antioxidant capacity of cereal and pasture species (Cartes et al. 2005; Mora et al. 2008; Cartes et al. 2011) and alleviated the Al-induced oxidative stress (Cartes et al. 2010). In cereals, we have increased Se uptake by inoculating them with Se-tolerant rhizobacteria with a capacity to reduce inorganic Se to elemental Se (Acuña et al. 2013). An increase in Se content in wheat grains was also obtained by co-inoculation of rhizobacteria and arbuscular mycorrhizal fungi (Durán et al. 2013). These studies have demonstrated a great potential in the use of soil microorganisms as a biofortification strategy in cereals grown in Chilean Andisol. However, it is known that the rhizosphere is a complex and dynamic environment, and the effectiveness of inoculated rhizobacteria is strongly influenced by diverse factors, such as biotic (competition with other microorganisms, root exudates, etc.) and abiotic (pH, fertilization, soil type, etc.) factors. Endophytic bacteria have an ecological advantage over rhizobacteria because plant tissues offer their protection

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against environmental conditions (Pathak and Keharia 2013) and they have a stronger association with plants than rhizobacteria (Sturz et al. 1999; Reiter et al. 2002). Hence, endophytic bacteria frequently result in greater benefits to plants than plant-growth-promoting rhizobacteria (PGPR) in terms of plant nutrition, hormonal enhancement of root growth, and disease suppression ( Lodewyckx et al. 2002; Mastretta et al. 2006; Taghavi et al. 2007; Ryan et al. 2008). Therefore, endophytic bacteria are a promising alternative to rhizobacteria for plant inoculation, also having a potential use in environmental restoration (phytoremediation) and agriculture (e.g., soil-borne pathogen control) (Strobel et al. 1996; Zhang et al. 1999; Strobel and Daisy 2003; Babu et al. 2013). Andisols have a high incidence of soil-borne pathogen Gaeumannomyces graminis var. tritici causing take-all (white heads), which is considered the most severe wheat disease in southern Chile (Andrade et al. 2011). The control of take-all by chemical management is expensive and poorly effective at best (Sari et al. 2008). Our main objective was to isolate and select putative plantgrowth-promoting endophytic bacteria from different tissues of Se-supplemented wheat as a potential inoculum for dual purposes: biofortification and G. graminis biocontrol. We have also characterized the bacterial community composition in the rhizosphere using denaturing-gradient gel electrophoresis (PCR-DGGE) because it has been postulated that the main entry of endophytic bacteria is via roots (Reinhold-Hurek and Hurek 2011).

Material and methods Isolation of endophytic bacteria from wheat plants grown under field conditions Wheat seeds (cultivars Fritz and Puelche) were coated with sodium selenite at 2 g kg−1 seed equivalent to 400 g Se ha−1 (+ Se), and control plants were treated in the same way but without sodium selenite (−Se). Plants were sown in Freire series of Andisol (CIREN 2002) and grown until maturity at Maquehue Experimental Station of La Frontera University (38° 50′ S, 72° 41′ W) located in the central valley of La Araucanía region in southern Chile. The soil has silt loam texture (0–20 cm) and bulk density varying from 0.82 to 1.32 g cm−3 with depth. The field experiment was set up in completely randomized block design with factorially arranged treatments (two cultivars × ± Se) with three replicates for each treatment. Representative plants and rhizosphere soil samples were collected from each plot stored at 4 °C and transported to the laboratory within 2 h. Roots, stems, leaves and spikes were separated, surfacesterilized by repeated immersion in 80 % v/v ethanol for 5 min and 4 %v/v NaOCl for 20 min, and then rinsed three

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times with sterile distilled water (Shimizu 2011). Tissue samples were macerated and homogenized in 1 mL of sterile saline solution (0.85 %v/v NaCl). One hundred microliters of homogenized tissue dilutions was spread onto the general media Luria-Bertani (LB) and potato dextrose (PD) agar plates and incubated at 30 °C for 4 days. Additionally, the efficacy of tissue surface sterilization was confirmed by spreading of last-run rinsing water onto LB and PD media as described above. Selenium tolerance of endophytic bacteria and plant growth-promoting traits Isolated endophytic bacteria were screened for Se tolerance. The minimum inhibitory concentration (MIC) of Se was determined on a medium with sucrose, minimal salts, and low phosphate (SLP; He et al. 2010) supplemented with sodium selenite (Na2SeO3 ×5H2O) at final concentration: 0, 5, 10, 20, 40, 60, 80, 100, 120, 140, 180, and 200 mM. Appropriate dilutions of fresh bacterial cultures were spread onto Sesupplemented agar plates and then incubated at 30 °C for 4 days. After incubation, colony growth on −Se and +Se plates was compared. We determined the following plant-growth-promoting (PGP) traits: (i) the production of phytohormone indoleacetic acid (IAA) and siderophores and (ii) the capacity to utilize insoluble organic and inorganic phosphorus (P) forms. The IAA production was determined at 535 nm using Salkowski’s reagent as described by Patten and Glick (2002). The siderophore production was evaluated on agar plates supplemented with chrome azurol S (CAS) reagent as described by Alexander and Zuberer (1991). The capacity to utilize P on agar was examined based on the appearance of clear zones around the colonies on National Botanical Research Institute’ phosphate (NBRIP) growth medium supplemented with tricalcium phosphate (Ca3[PO4] 2) (Nautiyal 1999) and phytase screening medium (PSM) containing sodium phytate (C6H6Na12O24P6 ×H2O) as a sole P source (Kerovuo et al. 1998). Halo formation was measured as follows: +++, very high capacity; ++, high capacity; +, normal capacity; and −, no capacity. Genotypic characterization of endophytic bacteria Genetic characterization of selected bacteria was based on partial sequencing of 16S rRNA gene. The 16S rRNA gene fragments were amplified by PCR with universal bacterial primers set 27f (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492r (5′-TAC GGY TAC CTT GTT ACG ACT T-3′) (Peace et al. 1994). After starting at 94 °C for 5 min, PCR amplification was carried out for 35 cycles at 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 2 min. The PCR products were purified and sequenced by Macrogen Inc., (Korea). The

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sequences were deposited under accession nos. from KF561857 to KF561872 and compared with those in the GenBank database.

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assays were repeated three times (n=12), and the values were given as means ± standard errors. Differences were considered significant when the P value was less than or equal to 0.01.

Endophytic bacteria as biocontrol of G. graminis in vitro Endophytic bacteria selected from Se-supplemented plants according to Se tolerance (50 mM) were assayed by a dualculture technique for their biocontrol against G. graminis var. tritici (Ggt) pathogen. Ggt was grown on PD agar plates at 25 °C for 1 week. Agar disks (4-mm diameter) containing Ggt were aseptically incised and transferred to the center of agar plates containing fresh LB/PD (1:1) media. Then, two drops (5 μL) of each selected endophytic bacteria suspension were taken from overnight LB cultures supplemented with sodium selenite (5 mM), washed threefold with NaCl (0.85 %), and placed on two diametric positions 2 cm from the agar disk containing the Ggt inoculum. Fungal mycelia growth was measured after incubation for 2, 5, and 7 days at 25 °C in the darkness as described by Liu et al. (2011). In addition, biocontrol assays with endophytic bacteria grown in LB without sodium selenite were also carried out. Agar plates inoculated with Ggt and two 5-μL drops of distilled sterile water were used as a negative control. As a positive control, Ggt was grown in PDA media supplemented with sodium selenite. Rhizobacterial community of wheat plant rhizosphere Total DNA was extracted from rhizosphere soils with UltraClean Soil DNA Isolation Kit (Mo Bio Laboratories, Inc., USA), and DNA fragments were amplified by touchdown PCR using specific primers set (EUBf933-GC and EUBr1387) for bacterial 16S rRNA gene (Iwamoto et al. 2000). The PCR products were run in 6 % (w/v) polyacrylamide gel with a 30–65 % gradient (urea and formamide) at 100 V for 12 h. The gel was then stained with SYBR Gold (Invitrogen Co., USA) for 30 min and photographed on an UV transilluminator. The PCR-DGGE banding profiles were analyzed by Phoretix 1D analysis software (TotalLab Ltd., UK). Based on the matrix obtained from Phoretix 1D analysis, the presence or absence and abundance of bacterial groups were analyzed by nonmetric multidimensional scaling (MDS) using SPSS statistical software (SPSS, Inc.) and used to estimate the bacterial diversity by Shannon-Wiener index as described by Yang et al. (2003).

Results Endophytic bacteria isolated from wheat plants grown under field conditions Our study revealed that tissues (roots, stems, and leaves) of both wheat cultivar harbor culturable endophytic bacteria with a variety of colony phenotypes. All endophytic isolates had a capacity to reduce Se from inorganic sodium selenite to elemental Se (Se0), visualized as red colonies on agar plates. Based on the MIC (Table 1), bacteria isolated from +Se wheat plants showed greater Se tolerance (60 to 180 mM) than those from −Se (10 to 100 mM). For biofortification purposes, our selection criteria were based on 50 mM of Se tolerance. Plant-growth-promoting traits of selected endophytic bacteria Around 67 % of endophytic isolates were selected from +Se and −Se mineralized phytate on PSM and solubilized tricalcium phosphate on NBRIP media based on halo formation around colonies. Enterobacter sp. EC5.2 showed the strongest capacity of phytate mineralization and P solubilization (Table 1). In general, Gram-positive strains did not show phytate mineralization and P solubilization on agar, except Bacillus sp. EC6. Gram-negative Acinetobacter sp. E6.2 also did not show phytate mineralization and P solubilization capability. Isolates from +Se plants showed high IAA production (up to 94 μg mL−1) compared with isolates from −Se plants (up to 35 μg mL−1). The highest IAA production was recorded for Klebsiella strains E2 (94 μg mL−1) and E8.2 (90 μg mL−1), whereas Bacillus sp. E8.1, Bacillus sp. EC8.2, and Paenibacillus sp. EC8.3 did not produce this phytohormone (Table 1). The bacterial strains isolated from +Se wheat plants that did not utilize P from organic and insoluble inorganic sources (i.e., all Gram-positive isolates and Gram-negative Acinetobacter sp. E6.2) also did not produce siderophores (Table 1), whereas all isolates from −Se wheat plants produced siderophores. Genotypic characterization

Statistical analysis Data were analyzed by a one-way analysis of variance (ANOVA), and comparisons were carried out for each pair with Tukey test by SPSS software (SPSS, Inc.). Biocontrol

Identification and phylogenetic affiliation of isolates based on partial sequencing of 16S rRNA genes are shown in Table 2. Around 1,350 bp of length of the fragment was amplified. Selected endophytic bacteria belonged to Proteobacteria (67 %) and Firmicutes (33 %) phylum.

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Table 1 Characteristics of putative endophytic bacteria tolerant to elevated Se content (50 mM) and isolated from tissues of wheat plants treated with (+Se) and without (−Se) Se Isolate

Cultivar Tissue Colony phenotype Shape

Color

Size

Margin

Gram MIC (mM) IAA (μg mL−1)

PM PS

Siderophore production

+Se Klebsiella sp. E1 Klebsiella sp. E2 Klebsiella sp. E3.1 Klebsiella sp. E4 Bacillus sp. E5 Bacillus sp. E6.1 Acinetobacter sp. E6.2 Klebsiella sp. E6.3 Bacillus sp. E8.1 Klebsiella sp. E8.2 −Se Enterobacter sp. EC4 Enterobacter sp. EC5.2 Bacillus sp. EC6 Bacillus sp. EC8.2 Paenibacillus sp. EC8.3

Fritz Fritz Fritz Fritz Puelche Puelche Puelche Puelche Puelche Puelche

Root Stem Leaf Spike Root Stem Stem Stem Spike Spike

Oval Oval Oval Oval Irregular Irregular Irregular Oval Irregular Oval

Cream Cream Cream Cream Cream Cream Cream Cream Cream Cream

Small Small Small Small Large Large Moderate Small Large Small

Entire Entire Entire Entire Undulate Undulate Filiform Entire Undulate Entire

− − − − + + − − + −

140 180 80 120 120 120 60 60 80 80

69.2 94.1 77.2 30.5 38.2 39.9 37.9 38.7 n.d. 89.6

+ + + + − − − + − +

+ + + + − − − + − +

+ + + + − − − + − +

Fritz Puelche Puelche Puelche Puelche

Spike Root Stem Spike Spike

Oval Oval Irregular Irregular Irregular

Cream Cream Cream Cream Cream

Small Small Large Large Large

Entire Entire Undulate Undulate Undulate

− − + + −

50 100 80 80 100

34.2 34.7 28.5 n.d. n.d.

++ +++ + − −

+ +++ ++ − −

+ + + + +

MIC minimum inhibitory concentration, IAA indole-3-acetic acid, PM mineralization of phytate on PSM, PS solubilization of phosphate on NBRIP, n.d. not detected, positive sign positive reaction, negative sign negative reaction

By considering the tree of 16S rRNA gene sequences from GenBank database (Fig. 1), our isolate showed similarity with other reported endophytic strains and with rhizobacteria isolated from the same Andisol (Acuña et al. 2013).

Biocontrol of G. graminis in vitro The most of selected endophytic strains inhibited mycelia growth 7 days after inoculation (Table 3). The fungal

Table 2 Phylogenetic affiliation of putative endophytic bacteria Isolate

Closest relatives or cloned sequences (accession no.)a

Similarity (%)

Accession no.

Klebsiella sp. E1 Klebsiella sp. E2 Klebsiella sp. E3.1 Klebsiella sp. E4 Bacillus sp. E5 Bacillus sp. E6.1 Acinetobacter sp. E6.2 Klebsiella sp. E6.3 Bacillus sp. E8.1 Klebsiella sp. E8.2 Enterobacter sp. EC4 Enterobacter sp. EC5.2 Bacillus sp. EC6 Bacillus sp. EC8.2 Paenibacillus sp. EC8.3

Klebsiella oxytoca endophytic bacteria from maize (GQ496663) Klebsiella oxytoca endophytic bacteria from maize (GQ496663) Klebsiella oxytoca endophytic bacteria from maize (GQ496663) Klebsiella oxytoca PGPR from wheat associated microflora (KF054939) Bacillus sp. nectar-associated bacteria in wild plant communities (JN872501) Bacillus axarquiens novel endophytic bacteria isolated from sugarcane (KC915228) Acinetobater sp. endophytic bacteria isolated from ethnomedicinal plants (JQ074047) Klebsiella oxytoca isolated from wheat rhizosphere (KF054939) Bacillus sp. nectar-associated bacteria in wild plant communities (JN872501) Klebsiella oxytoca endophytic bacteria from maize (GQ496663) Enterobacter ludwigii endophytic bacteria from Panax notoginseng (JN700140) Enterobacter ludwigii endophytic bacteria from peanut plants (JQ308612) Bacillus axarquiensis endophytic bacteria from sugarcane (KC915228) Bacillus sp. PGPR from rice (KF358286) Bacillus mycoides endophytic bacteria from tobacco (JN999844)

99 99 99 99 99 98 99 99 99 99 96 88 99 99 99

KF561864 KF561865 KF561866 KF561867 KF561868 KF561869 KF561870 KF561860 KF561871 KF561872 KF561857 KF561858 KF561859 KF561862 KF561863

a

Based on partial sequencing of 16S rRNA gene and comparison with sequences found in GenBank by using BLASTN

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Fig. 1 Phylogenetic tree showing the affiliation of the selected endophytic bacteria isolated from wheat plants with (black circle) and without (white circle) Se treatment. The neighbor-joining tree was constructed with representative 16S rRNA gene sequences of culturable endophytic bacteria (white triangle) and selenium-tolerant rhizobacteria isolated from similar study area (black triangle). The bar indicates 2 % sequence divergence; a bootstrap analysis was performed with 1,000 runs. The accession numbers are in parentheses

inhibition was around 100 % for Acinetobacter sp. E6.2, 50 % for Bacillus sp. E8.1, and 30 % for Klebsiella sp. E1 and Bacillus sp. E5. In contrast, Klebsiella sp. E3.1 and E4 did not show fungal inhibition. Similar effect was noted in bacteria grown in sodium selenite as the biosynthetized selenium form. It is important to underline that sodium selenite as inorganic form can inhibit the fungal growth at minimal doses (Fig. 2d).

analysis confirmed this result showing an average value of 1.35 for the Se-biofortified Fritz cultivar, whereas for Puelche cultivar with and without Se and Fritz cultivar without Se, the average value was 1.2.

Rhizobacterial communities associated with wheat rhizosphere

Fifteen endophytic strains were isolated from Sesupplemented and control wheat plants belonging to Bacillus, Paenibacillus, Klebsiella, Enterobacter, and Acinetobacter genera and selected according to high Se tolerance (50 mM). Neighbor-joining tree of 16S rRNA gene sequences from GenBank database showed that our isolates have genetic similarities with respect to previously reported endophytic strains and with rhizospheric bacteria isolated from the same Andisol from southern Chile by Acuña et al. (2013). Thus, these endophytic strains can be considered as facultatively

The composition of rhizobacterial communities associated with sampled wheat plants is shown in Fig. 3. According to dendrogram and MSD analysis, wheat cultivar Fritz from the −Se treatment showed a major diversity compared with cultivar Puelche and cultivar Fritz grown in the +Se treatment. Interestingly, rhizobacterial communities of cultivar Puelche with and without Se were similar. The Shannon-Wiener index

Discussion

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Table 3 Inhibition of Gaeumannomyces graminis var. tritici by selected endophytic strains after 2, 5, and 7 days of incubation Isolate

Diameter of fungal spread (cm) −Se

+Se

Klebsiella sp. E1 Klebsiella sp. E2 Klebsiella sp. E3.1 Klebsiella sp. E4 Bacillus sp. E5 Bacillus sp. E6.1 Acinetobacter sp. E6.2 Bacillus sp. E8.1 Control

2d

5d

7d

2d

5d

7d

0.43±0.11 abc 0.63±0.10 a 0.58±0.09 a 0.54±0.07 ab 0.27±0.12 abc 0.44±0.12 abc 0.10±0.04 bc 0.01±0.00 c 0.60±0.06 a

1.70±0.20 ab 2.27±0.15 a 2.28±0.08 a 2.16±0.14 ab 1.48±0.19 b 1.96±0.19 ab 0.00±0.00 c 0.68±0.08 c 2.27±0.08 a

2.92±0.23 b 3.59±0.21 ab 3.67±0.13 a 3.63±0.14 a 2.92±0.21 b 3.50±0.15 ab 0.00±0.00 d 2.11±0.09 c 4.00±0.00 a

0.04±0.02 c 0.25±0.09 abc 0.62±0.08 a 0.19±0.08 bc 0.19±0.09 bc 0.53±0.09 ab 0.44±0.07 abc 0.39±0.09 abc 0.60±0.06 ab

1.05±0.07 b 1.50±0.18 abc 2.18±0.11 ab 1.28±0.13 a 1.58±0.17 b 1.56±0.27 abc 0.00±0.00 c 1.43±0.23 bc 2.27±0.08 a

2.48±0.15 c 2.94±0.22 bc 3.75±0.12 ab 2.76±0.21 c 3.12±0.18 bc 3.10±0.27 bc 0.00±0.00 d 2.83±0.27 c 4.00±0.00 a

Values represent mean ± standard error (average of three repeats). Different letters in the column denote significant difference (P≤0.01, comparisons of means were carried out for each pair with Tukey test by using SPSS software (SPSS, Inc.) +Se isolate previously grown on media supplemented with sodium selenite, −Se isolate previously grown on media without sodium selenite

endophytes and probably may also live outside the plant tissues as rhizospheric bacteria (Lodewyckx et al. 2002). It should also be kept in mind that endophytes are better adapted to elevated Se concentration than rhizospheric bacteria, since rhizobacteria can only tolerate around 20 mM of sodium selenite only (Acuña et al. 2013), whereas our endophytic strains showed tolerance to 60 to 180 mM of Se. Similar tolerant values were reported by Hunter and Manter (2009) for Pseudomonas sp. (150 mM of Se). According to DGGE analysis and Shannon-Wiener index communities associated to Fritz, plants showed a higher diversity than those associated to Puelche control wheat plants. Also Siciliano et al. (1998) and Germida and Siciliano (2001) reported that composition of rhizospheric communities depends on wheat cultivar. In plants supplemented with Se, no significant differences were found. We can suggest that Fritz was affected by Se supplementation at doses of 400 g Se ha−1 considering the diminished microbial biodiversity in the treatment with selenium. Divergence in PGP traits between endophytic bacteria isolated from control or Se-supplemented treatments was observed. Most strains (67 %) were able to solubilize P in Fig. 2 Growth of Gaeumannomyces graminis var. tritici in the absence of endophytic bacteria (a) and in the presence of Acinetobacter sp. E6.2 (b) and Klebsiella sp. E3.1 (c) and in sodium-selenitesupplemented media (d). Arrows denote colonies grown on agar plate

NBRIP media with tricalcium phosphate (TCP). The TCP is the universal method for evaluating phosphate solubilizing bacteria in vitro conditions (Nautiyal 1999; Jorquera et al. 2008; Martínez-Viveros et al. 2010), but recently, it has been shown that it is inaccurate and the use of several phosphates has been suggested to determine the P solubilization (Bashan et al. 2013a, b). Siderophore production occurred from Bacillus strain from the control plants but not from Bacillus strain isolated from Se-supplemented plants. In contrast, strains isolated from Se-supplemented plant showed a major IAA production in comparison with control, suggesting that strains evolved survival mechanisms against stress conditions. Indeed, Kaya et al. (2013) reported that IAA plays a vital role in maintaining plant growth under stresses, such as those due to Se stress and tolerance (Wu and Huang 1991). Acinetobacter sp. (strain E6.2), Bacillus sp. (strain E8.1), Bacillus sp., and Klebsiella sp. (strain E5 and E1) inhibited the G. graminis mycelia growth in vitro (100, 50, and 30 %, respectively). Similarly, Liu et al. (2011) showed that endophytic Bacillus subtilis can successfully inhibit the development of G. graminis under field conditions. Endophytic bacteria can diminish the development of different

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Fig. 3 Dendrogram (left) and nonmetric multidimensional scaling (right) analysis of DGGE profiles (16S rRNA gene) from rhizobacterial communities of wheat plants (Fritz and Puelche cultivars) with and without Se supplementation

phytopathogens, i.e.,: Erwinia carotovora (Reiter et al. 2002), Botrytis cinerea (An and Ma 2005), Rhizoctonia solani (Cho et al. 2007), and Streptomyces scabies (Lin et al. 2012). Putative endophytic bacteria promoting plant growth could represent promising biocontrol agents due to their competition with pathogens in the ecological niche and their localization inside the plants and biofortification agents, being more effective than that rhizobacteria for enhancing Se uptake by plants. In addition, they are efficient than rhizospheric bacteria since apoplast is a less competitive environment than the rhizosphere (Reiter et al. 2002). Consequently, there may not be the need to select bacterial types with high levels of rhizosphere competence for a successful seed or root inoculation treatments (Sturz et al. 1999; Conn and Franco 2004). In addition, the abundance of rhizospheric bacterial populations declines rapidly (by orders of magnitude per week) before reaching an environmental equilibrium (Martínez-Viveros et al. 2010) and reduces the endophytic colonization and diversity (Conn and Franco 2004). To the best of our knowledge, this is the first report on endophytic bacteria being used for selenium biofortification strategies and crop protection. In the future studies, these potential purposes should be explored under field conditions.

Conclusions We could suggest Acinetobacter, Bacillus, and Klebsiella endophytic strains as inoculant for Se biofortification technologies due to (i) their elevated Se tolerance, (ii) their promoting effects on plant growth, and (iii) their protection against G. graminis, the most important pathogen of cereal crop grown in Andisol from southern Chile. Our results suggest that selected endophytic strains can potentially be used as microbial inoculants for biofortification and biocontrol against G. graminis.

Acknowledgments This study was supported by the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) postdoctoral scholarship N° 3130542 and CONICYT regular project N° 1100625 by the Chilean Government.

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