Identification and characterization of the part of the bacterial community associated with field-grown tall fescue (Festuca arundinacea) cv. SFRO Don Tomás in Uruguay

Abstract

The aims of this study were to isolate, characterize and identify the native culturable putative endophytic bacterial community associated with tall fescue (Festuca arundinacea) cv. SFRO Don Tomás, cultivated in Uruguay, and to study the effects of inoculation on cv. SFRO Don Tomás and the commercial cv. Tacuabé. A total of 342 isolates were collected from surface-sterilized roots, stems and seeds of healthy cv. SFRO Don Tomás. The functional ability of the isolates to produce indole, to solubilize minerals (P, Fe, K) and to biologically fix molecular nitrogen (N₂) was determined. Several infection traits, such as the ability to produce proteases, peroxidases, cellulases and hemicellulases, were identified in the isolates. Selected bacterial isolates were identified by 16S rRNA sequencing and shown to belong to a broad spectrum of genera, including Bacillus, Microbacterium, Curtobacterium, Streptomyces, Acidovorax, Variovorax, Acinetobacter, Pseudomonas, Pantoea, Rhanella and Xanthomonas. Plant growth promotion assays shown that ten isolates were able to promote the growth of cv. SFRO Don Tomás under gnotobiotic conditions, thereby highlighting the potential of these isolates in biotechnological applications as inoculant for this cultivar which is highly adapted to dry and cold seasons.

Introduction

Tall fescue (Festuca arundinacea) is an important cool-season forage grass native to North Africa and Northern Europe which was introduced to several parts of the world, such as the USA, Argentina, Uruguay, Australia and New Zealand, for livestock feed (Hoveland 2010). In Uruguay, whose economy mainly depends on livestock and meat export, tall fescue is the most widely cultivated cool-season grass, grown on 70 % of the total cultivated pasture area (500,000 ha), due to its higher productivity and persistence compared to perennial ryegrass (Lolium perenne) (Milne 2010). Under optimal hydric growth conditions tall fescue has a high dry weight yield and is perennial during the winter. Moreover, in Uruguay, its high persistence and productivity is associated with excellent drought and cold resistance, as well as a high competitive capacity relative to one of the principal Uruguayan undergrowths, Bermuda grass (Cynodon dactylon) (Carambula 2000). Tall fescue propagation is by seed, and the plants display low vigor in the first winter, which makes the plant suitable for sowing in mixtures with other, less competitive forages, such as the legumes Trifolium repens and Lotus corniculatus (Milne 2010). Tall fescue is important in rotation systems due to its contribution to the productivity persistence of the prairie, in complementation with legume crops, leading to an improvement of soil physicochemical properties. This type of agricultural system allows a more sustainable use of the soil and helps control erosion as well as maintain organic carbon levels in the soil (García Préchac et al. 2002).

Bacterial endophytes are defined as those bacteria which can be detected at a particular moment within the tissues of apparently healthy plants without producing symptoms (Schulz et al. 2006). In contrast with the well-studied symbiotic or pathogenic systems, little is known about the molecular basis of endophyte–plant interactions. Nevertheless, there is an abundance of evidence which testifies their beneficial effects on plant growth promotion (PGP) in several host plants (Hardoim et al. 2008; Ryan et al. 2008; Compant et al. 2010; Mei and Flinn 2010). Endophytic PGP can be achieved directly by biofertilization [fixation of molecular nitrogen (N₂); solubilization of phosphorus (P), potassium (K) and iron (Fe)] by the production and regulation of phytohormones or indirectly by stress tolerance, pathogen biocontrol or the induction of systemic resistance in the plants (Mercado-Blanco and Lugtenberg 2014). Taking this into account, the biotechnological exploitation of the endophyte–host interaction might play a significant role in improving the sustainability of agricultural systems.

During a long dry season in 2008, technicians from the Rural Promotion Society Ortiz (SFRO) identified a green, healthy and vigorous naturalized population of tall fescue alongside Route 8 km 145 in Lavalleja State, Uruguay (34°17′26.60″ S, 54°59′14.77″ W; 123 m a.s.l.). This population was harvested and multiplied and its productive behavior tested under commercial conditions, i.e. a sheep farm, where it showed an excellent response. This led to its registration as a cultivar under the name SFRO Don Tomás, and it has become an excellent model for studying the native culturable bacterial endophytic community associated with tall fescue. The application of native plant growth-promoting bacteria (PGPB) might improve yields by direct PGP or by biocontrol and/or by increasing plant tolerance to stress (Mercado-Blanco and Lugtenberg 2014). In this context and with the general aim of contributing to the economic and environmental sustainability of the tall fescue cv. SFRO Don Tomás by using naturally associated PGPB, the aims of this study were: (1) to obtain a large collection of culturable putative endophytic bacteria associated with tall fescue cv. SFRO Don Tomás, (2) to characterize this collection based on PGP and infection traits, and thus to identify isolates of interest for agricultural use and (3) to study the inoculation effects of selected isolates on the growth of tall fescue cv. SFRO Don Tomás and the commercial cv. Tacuabé. The data obtained will contribute to future research toward the development of an inoculant based on native endophytic PGPB for tall fescue cv. SFRO Don Tomás.

Material and methods

Isolation of putative endophytic bacteria from tall fescue cv. SFRO Don Tomás

Putative endophytes associated with tall fescue cv. Don Tomás were isolated from the seeds, roots and aerial parts of adult plants. Healthy plants were collected from three sites (A–C) in the Lavalleja Department in the eastern region of Uruguay (A: 34°18′30.5″S, 55°24′1.4″W, 125 m a.s.l.; B: 34°17′26.60″S, 54°59′14.77″W, 123 m a.s.l.; C: 34°14′54.0″S, 55°23′33.5″W, 120 m a.s.l.), where no fertilization had been applied for the preceding 4 years but where good productivity had been still registered over that period. The soil profiles of sites A and B were as follows: A (pH 5.7, 13 % sand, 54 % silt, 33 % clay, 2.5 % organic matter, total N 0.28 %), B (pH 5.8, 30 % sand, 67 % silt, 13 % clay, 2.9 % organic matter, total N 0.55 %). The protocol employed for bacterial isolation was as reported by Mareque et al. (2015) with slight modifications in the incubation times and the culture media employed. Seeds and plant tissues (root and stems) were surface-disinfected by incubation in 5 % HClO₄ for 45 min and in 4 % HClO₄ for 15 min, respectively. Dilutions of the suspension obtained were inoculated onto agar plates containing Tryptic Soy Agar (TSA; Difco Laboratories, Detroit, MI) and nitrogen-free combined carbon (NFCC) medium (Mirza and Rodrigues 2012) respectively, and in vials containing NFCC semisolid medium. NFCC plates were incubated at 30 °C under microaerobic conditions using the Microbiology Anaerocult® A Mini kit (Merck KGaA, Darmstadt, Germany) for 20 days, while TSA plates and NFCC vials were incubated under aerobic conditions at the same temperature. The isolates obtained were replicated twice in NFCC medium followed by two replications in TSA medium. Individual colonies were classified in TSA medium according their morphological features. This procedure was carried out with the aim of isolating both diazotrophic and heterotrophic bacteria.

The bacterial abundance in the roots and the aerial parts of plants collected from sites A and B was determined by counting the colony forming units per gram of fresh tissue (CFU g⁻¹) in TY (tryptone yeast extract) plates.

Screening of the bacterial collection for biofertilization and plant interaction traits

The entire bacterial collection was screened for putatively diazotrophic isolates by nifH-targeted PCR amplification using the primers PolF and PolR (Poly et al. 2001). The final reaction mixture (25 μl) consisted of 2.5 μl 10X Taq reaction buffer (Fermentas, Thermo Fisher Scientific, Waltham, MA) 3.0 mM MgCl₂, 0.16 mM dNTPs, 0.8 μM of both set of primers, 0.5 U Taq polymerase (Fermentas), 4 % bovine serum albumin and 4.0 μl of a cell lysate template and was amplified as described by Taulé et al. (2012) and Mareque et al. (2015). The ability to fix N₂ was also tested in those isolates, which harbored the nifH gene, in vials containing LGI and NFCC, N-free semisolid media (Reis et al. 1994; Mirza and Rodrigues 2012). The vials were incubated at 30ºC for up to 7 days, and those that showed a growth pellicle were replicated into a new fresh vial containing the same medium (Baldani et al. 2014). Those isolates that were available to grow as a pellicle in the semisolid N-free media and which harbored the nifH genes were considered to be diazotrophs.

Siderophore production was assayed according to Schwyn and Neilands (1987) using plates containing chromeazurol (CAS) medium. Plates were incubated for 72 h at 30 °C. Siderophore production was indicated by the presence of a yellow halo around the colony (positive test result).

The method described by Sylvester-Bradley et al. (1982) was used to identify isolates able to solubilize phosphates. Isolates were grown on plates containing GL medium (10 g glucose, 2 g yeast extract, 5 g K₂HPO₄, 10 g CaCl₂, 15 g agar) and incubated for 72 h at 30 °C. Phosphate-solubilizing ability was indicated by the presence of a translucent halo around the colony (positive test result).

For the detection of K-solubilizing isolates, the isolated bacteria were grown on plates containing Aleksandrov medium (10 g sucrose, 1.5 g K₂HPO₄, 0.5 g MgSO₄⋅7H₂O, 1 g CaCO₃, 15 g of agar; Avakyan et al. 1986) and incubated at 30 °C. K-solubilizing ability was indicated by the presence of a translucent halo around the colony (positive test result).

The colorimetric method described by Sarwar and Kremer (1995) was employed to test the entire collection of isolates with the aim of detecting the production of indole-3-acetic acid (IAA) as described previously (Taulé et al. 2012).

Endoglucanase and hemicellulase activities were screened by culturing the isolates on solid TSA culture media supplemented with 0.2 % carboxymethyl cellulose or 0.5 % Avicel, respectively (Kim et al. 2008). Positive strains were determined by degradation halos around each colony.

Protease activity was evaluated in plates containing TSA medium supplemented with 5 % skimmed milk. Strains were considered to possess protease activity when a translucent halo was observed around the colonies (Martinez-Rosales and Castro-Sowinsky 2011).

For the determination of peroxidase activity, we grew strains in plates containing TSA medium supplemented with 250 mg l⁻¹ of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS; Sigma-Aldrich, St. Louis, MO); for the determination of manganese (Mn)-peroxidases, the strains were grown on TSA medium with 250 mg l⁻¹ ABTS + 100 mg l⁻¹ MnCl₂·4H₂O (Sack et al. 1997). Strains were considered to possess peroxidase activity when the color of the colonies turned a dark green or brown, respectively.

Biofilm formation was screened in 96-well plates using the crystal violet method (Peeters et al. 2008). Each well containing 200 μl TSB medium was inoculated with each isolate at a final concentration of 1–2 × 10⁸ cells ml⁻¹ isolate. After incubation for 48 h at 30 °C without agitation, the supernatant was removed and the wells were washed with phosphate buffered saline (PBS), followed by staining with a 0.1 % CV solution for 20 min. The excess CV was removed by washing the plates with PBS, and the bound CV was solubilized with 95 % EtOH. The absorbance of the suspension was measured at 570 nm.

All determinations were performed in triplicate.

Physiological features of the bacterial collection

With the aim of determining the capacity of the isolates to grow on different C and N sources, we grew the entire bacterial collection on plates containing LGI medium supplemented with the C or N sources to be tested; these were provided at the same final concentrations as reported in the original protocol (Cavalcante and Dobereiner 1988). The C sources analyzed were maltose, mannitol, glucose, sucrose, malate, fructose, lactose, glycerine, pyruvic acid, unrefined sugar, ethanol and vinasse (byproduct generated during the fermentation of cane molasses). The N sources tested were (NH₄)₂SO₄, KNO₃, NH₄Cl, L-tyrosine, L-asparagine, L-glutamic acid and urea. All determinations were done in triplicate.

16S rRNA amplification, sequencing and phylogenetic analysis

Selected isolates were subject to 16S rRNA amplification and sequencing as described by Taulé et al. (2012). The 16S rRNA sequences were deposited in GenBank under the following accession numbers: KP704407–KP704439.

In the phylogenetic studies, the quality of the sequences obtained was checked manually and assembled using the software DNA Baser Sequence Assembler v3.× (2010) (http://www.dnabaser.com). The consensus sequences were aligned using Sina Web Aligner (Pruesse et al. 2007), imported into the ARB software package v 6.0.2 (Ludwig et al. 2004) and added to the database. Phylogenetic trees were generated using the neighbor-joining algorithm with 1000 bootstrap replicates.

PGP of tall fescue cvs. SFRO Don Tómas and Tacuabé under gnotobiotic conditions

Seeds of tall fescue cvs. Don Tomás and Tacuabé were surface-sterilized as described above and placed onto 0.8 % water agar plates at room temperature for 5 days. Two seedlings were then placed into each plant growth tube containing 20 ml of liquid Jensen medium (Vincent 1970). The plants were inoculated with a cell suspension of each isolate to be tested at a final concentration of 1.0 × 10⁸ cells ml⁻¹; there were eight replicates for each treatment. Plants without inoculation or chemical fertilization were used as negative controls, and a reference control without inoculation but with the addition of 0.05 % KNO₃ was employed since all the isolates tested were putative diazotrophs. Plants were maintained at 26 °C under a photoperiod of 16/8 h light/darkness in a growth room. The plants were harvested at 1-month post inoculation and dried at 60 °C until a constant weight was reached, at which time the total dry weight was determined.

Statistical analysis

For the statistical analyses, we performed an analysis of variance on parametric data using the SPSS for Windows v20.0 (IBM Corp., Armonk, NY) software program. When a significant difference could be confirmed, the treatment means were compared against the negative control using Dunnett’s test. For nonparametric data we performed the Krustal–Wallis test. When a significant difference could be confirmed, the treatment means were compared against the negative control using the Mann–Whitman test. All significant differences between treatments were expressed as p-values at the 0.1, 0.05 and 0.01 levels.

Results

Isolation and characterization of putative endophytic bacteria associated with tall fescue cv. SFRO Don Tomás

A collection of 342 bacterial isolates associated with the seeds (6 isolates), roots (192) and aerial parts (144) of healthy tall fescue cv. SFRO Don Tomás plants was obtained. Because they were isolated from surface-sterilized material, we considered these to be putative endophytes unless proven otherwise by microscopy. Tall healthy fescue plants were collected from a crop region which had not been treated with chemical fertilization and where no tall fescue toxicity symptoms had been reported. In all, 179, 74 and 89 bacterial isolates were obtained using TSA, NFCC solid and NFCC semisolid media, respectively. The bacterial population from the inner plant tissues was enumerated in samples collected from sites A and B. In both cases, bacteria were more abundant in the roots (1 × 10⁶ and 1 × 10⁵ CFU g⁻¹ for sites A and B, respectively) than in the aerial parts (1 × 10⁵ and 1 × 10³ CFU g⁻¹ for sites A and B, respectively). Additionally, for both plant parts, bacterial abundance was higher at site A (1 × 10⁶ and 1 × 10⁵ for roots and aerial part, respectively) than at site B (1 × 10⁵ and 1 × 10³ for roots and aerial part, respectively).

PGP features, such as the ability to fix N₂, solubilize inorganic P, K and produce siderophores and IAA, was studied in the entire collection. Results from selected isolates, including their in vitro plant growth promotion and plant infection traits as well as their colony morphology, are shown in Table 1.

Table 1 Plant growth promotion features of selected putative endophytic bacterial isolates from tall fescue (Festuca arundinacea) cv. SFRO Don Tomás

Of the 342 isolates tested, 45 putative diazotrophic isolates were detected by PCR amplification of the nifH gene. All of these were grown in vials containing LGI or NFCC N-free semisolid medium, resulting in 42 isolates capable of producing a growth pellicle. The production of the growth pellicle allowed them to be considered as potential diazotrophs (12.3 % of the collection). From this group of 42 potential diazotrophic isolates 20, 14 and 8 were isolated from TSA, NFCC (microaerobic) and NFCC (semisolid) culture media, respectively. In addition, 27 and 33 isolates (of the total 342 isolates) were able to produce IAA and siderophores, respectively, 36 isolates were able to solubilize phosphate and one isolate was able to to solubilize K.

The bacterial collection was also screened for the presence of plant infection traits, including hemicellulase, cellulase, protease and peroxidase activities, as well as biofilm formation. No isolates with hemicellulase, cellulase or peroxidase activity were present in the collection, but 45 and 140 isolates demonstrated protease activity and biofilm-forming ability, respectively.

The entire bacterial collection was also evaluated for its ability to grow on different C and N sources (Table S1, electronic supplementary material). In terms of growing on a sole C source, 5 % of the isolates grew on sugarcane vinasse, 19 % on ethanol, 34 % on malic acid, 46 % on lactose and 60–81 % on fructose, pyruvic acid, sucrose, crystal sugar, glycerine, maltose, mannitol and glucose. In terms of growing on a sole N source, 50 % of the isolates grew on (NH₄)₂SO_4, 58 % on NH₄Cl and 83–88 % on KNO₃, urea, L-tyrosine, L-glutamic acid and L-asparagine.

Identification and phylogenetic analysis of tall fescue-associated isolates based on their partial 16S rRNA sequences

In the identification and phylogenetic analyses, we performed 16S rRNA sequencing of selected isolates and analyzed the sequence data by BLAST searches against the NCBI database. Isolates were selected for 16S rRNA sequencing on the basis of their in vitro PGP and plant infection traits. The nearest match from the GenBank database revealed a broad spectrum of isolates, including bacteria belonging to the phyla Firmicutes (Bacillus) and Actinobacteria (Microbacterium, Curtobacterium, Streptomyces) and a high number of isolates in the Proteobacteria. Among the latter, we identified isolates from the Betaproteobacteria (Acidovorax, Variovorax) and Gammaproteobacteria classes (Acinetobacter, Pseudomonas, Pantoea, Rhanella, Lelliottia, Xhantomonas) (Table 2).

Table 2 16S rRNA nucleotide sequence similarities of selected putative endophytic bacterial isolates from the Uruguayan tall fescue cv. SFRO Don Tomás

A phylogenetic tree was constructed based on the 16S rRNA sequences of 33 isolates (Fig. 1). Within the Proteobacteria, 23 isolates belonged to the class Gammaproteobacteria (Fig. 1; Table 2), of which isolates UYFA10, UYFA15, UYFA110, UYFA190 and UYFA198 were placed in the genus Pantoea and grouped in a well-supported cluster (Fig. 1), although only isolate UYFA110 was closely related to the reference strain P. ananatis AY530795 (Fig. 1). In a separate and well-supported branch, isolate UYFA298, which has 99 % identity to Lelliottia amnigena JCM1237 (Table 2), was closely related to the reference strain Enterobacter amnigenus AB004749. The isolates within the genus Rhanella (UYFA288, UYFA289, UYFA330 and UYFA337) formed a well-supported cluster that was closely related to the reference strain Rhanella aquatilis AJ233426 (Fig. 1). Acinetobacter spp. were well represented in the collection and formed a cluster with two branches. In the first branch, isolates UYFA01, UYFA06, UYFA24 and UYFA39 grouped close to the reference strain Acinetobacter calcoaceticus AJ888983, while in the second one, isolate UYFA02 grouped alone with non-reference strains (Fig. 1). Analysis of the 16S rRNA sequences of isolates UYFA95, UYFA113, UYFA214 and UYFA249 showed that these isolates belonged to the genus Pseudomonas (Table 2; Fig. 1) and that they formed two well-supported branches. Among these isolates, only isolate UYFA113 grouped with the reference strain Pseudomonas seleniipraecipitans FJ422810 (Fig. 1).

Fig. 1

Neighbor-joining phylogenetic tree based on bacterial 16S rRNA sequences of the representative isolates. The tree shows the phylogenetic affiliation of 30 partial 16S rRNA sequences of endophytic bacteria isolated from Tall fescue (Festuca arundinacea). Numbers at branches Bootstrap values of >50 % from 1000 replicates. Thermanaeromonas toyohensis ^T AB062280 was used as an outgroup. Scale bar Number of nucleotide substitutions per site

From the Gammaproteobacteria, two isolates in the genus Xanthomonas, UYFA105 and UYFA215, with 99 and 100 % identity with X. translucens XT2 (Table 2), clustered in a branch that was distantly related to the reference strains.

The Betaproteobacteria were represented in the phylogenetic tree by isolates UYFA22 and UYFA23. Isolate UYFA22 belonged to genus Variovorax (Table 2) and clustered close to the reference strain V. paradoxus AJ420329, while isolate UYFA23, which belonged to genus Acidovorax, was closely related to the reference strain Acidovorax avenae subsp. avenae, DQ360414 (Fig. 1).

With respect to the Firmicutes, analysis of the 16S rRNA sequences of isolates UYFA152 and UYFA154 showed that these belonged to genus Bacillus and were grouped in a well-supported cluster. Whereas isolate UYFA152 grouped with the reference strains Bacillus safensis AF234854 and B. pumilis AY876289, isolate UYFA154 was distant from any reference strain (Fig. 1). In addition, two isolates, UYFA153 and UYFA156, which have an identity of 99 and 100 % with Streptomyces sampsonii NRRL and B12325, respectively (Table 2), were clustered in a separate branch distantly related to the reference strains (Fig. 1).

The Actinobacteria were also well represented in the collection (Table 2), and the phylogenetic tree based on 16S rRNA sequences showed that isolates from this phylum grouped in a single cluster (Fig. 1). The genus Microbacterium was represented by isolates UYFA26, UYFA61, UYFA68 and UYFA09. The latter isolate did not group with any reference strains, but the remaining isolates grouped together in a cluster closely related to the reference strain M. binotii EF567306 (Fig. 1). Additionally, two isolates of Actinobacteria with 100 % identity with Curtobacterium flaccumfaciens LMG 3645 were identified in the collection (Table 2). Of these, isolate UYFA155 clustered closely with the type strain C. flaccumfaciens AJ312209, while isolate UYFA157 grouped in another branch distant from any reference strains (Fig. 1).

PGP of tall fescue cv. Don Tomás and Tacuabé inoculated with selected bacterial isolates

Twenty-eight isolates were selected on the basis of their in vitro PGP and plant infection traits, as well as on their generic identity (Tables 1, 2). These selected isolates were tested as inoculants on tall fescue cv. SFRO Don Tomás and Tacuabé under gnotobiotic conditions. At 1 month post inoculation, plantlets were harvested and their stem heights and total dry weights determined (Table 3).

Table 3 Effects of inoculation with putative bacterial endophytes on the growth of tall fescue cv. SFRO Don Tomás under gnotobiotic conditions

In the case of cv. SFRO Don Tomás, results from the statistical analysis showed that both parameters analyzed were significantly higher than the negative control for the isolates UYFA61, UYFA156, UYFA157 and UYFA215. Additionally, in isolates UYFA09, UYFA10, UYFA15, UYFA22, UYFA26 and UYFA249, the stem heights were significantly higher than those of the negative control, while plant dry weight was significantly higher for the isolate UYFA68 than for the negative control (Table 3).

In contrast to cv. SFRO Don Tomás, in the case of the commercial cv. Tacuabé, only isolates UYFA289 and UYFA337 were able to promote aerial dry weight and stem height, respectively (Table 4).

Table 4 Effects of inoculation with putative bacterial endophytes on the growth of tall fescue cv. Tacuabé under gnotobiotic conditions

Discussion

A variety of bacteria with PGP traits were found to be present in the roots, stems and seeds of tall fescue cv. SFRO Don Tomás

A collection of 342 bacterial isolates was obtained from surface-desinfected roots, stems and seeds of tall fescue cv. SFRO Don Tomás. The bacterial population was more numerous in the roots than in the aerial parts, which is in agreement with reports that roots are the most common colonization sites for endophytic bacteria and that only a small proportion of endophytic bacteria are able to colonize the internal aerial tissues (Mercado-Blanco and Lugtenberg 2014).

Among all of the bacterial isolates collected, 45 harbored the nifH gene and were able to grow on NFCC N-free semisolid medium, while 42 were also able to grow on NFCC N-free semisolid medium under microaerophilic conditions; these isolates were therefore considered to be diazotrophic. In addition, numerous isolates from the collection had various PGP traits, including the ability to produce IAA and solubilize P, K and Fe; interestingly, however, all were negative for the plant infections traits evaluated, with the exception of biofilm formation. The roles of IAA production and the ability to solubilize P and Fe in beneficial bacteria–plant interactions and growth promotion have been well reported (Rodríguez and Fraga 1999; Shokri and Emtiazi 2010; Ahmed and Holmström 2014). Additionally, our bacterial collection was highly versatile in the use of different C and N sources. Taken together, these data highlight the biotechnological potential of the bacterial collection in the production of a bioinoculant for cv. SFRO Don Tomás.

Our phylogenetic analysis of selected isolates revealed a broad range of genera in the collection, including Bacillus from the Firmicutes, Microbacterium, Curtobacterium and Streptomyces from the Actinobacteria, Acidovorax and Variovorax from the Betaproteobacteria, as well as a large number of isolates belonging to the Gammaproteobacteria, such as Acinetobacter, Pseudomonas, Pantoea, Rhanella, Lelliottia and Xanthomonas. These results are in contrast with the composition of a collection of bacteria associated with tall fescue reported in New Zealand, where the identified isolates were mostly members of the Gammaproteobacteria (Monk et al. 2009).

Most of the genera identified in our study have been previously reported as being associated with several agronomical crops, including phytopathogens such as Acidovorax oryzae, Xanthomonas albilineans, X. sacchari. However, the initial plant material which we collected for bacterial isolation had no disease symptoms, which is an essential prerequisite for endophytic bacteria (Hallmann et al. 1997; Rosenblueth and Martínez-Romero 2006). Within the Gammaproteobacteria, isolates of the genera Pantoea, Acinetobacter, Rhanella and Pseudomonas have been previously reported as being associated with and/or endophytic within several poaceous plants, including sweet sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), maize (Zea mays), rice (Oryza sativa), grapevine (Vitis vinifera), canola (Brassica napus) and others (Bell et al. 1995; Misko and Germida 2002; Hallmann and Berg 2006; Rosenblueth and Martínez-Romero 2006; Taulé et al. 2012; Mareque et al. 2015). Of particular significance is that Pseudomonas and Rhanella isolates have been previously reported as being associated with tall fescue (Monk et al. 2009).

With respect to the Betaproteobacteria, the only two isolates identified in this study belonged to the genera Variovorax (UYFA22) and Acidovorax (UYFA23). Isolates from these genera have been reported to be endophytic and plant-associated bacteria with canola (Germida et al. 1998; Graner et al. 2003).

The Actinobacteria in our collection were represented by isolates belonging to the genera Microbacterium, Curtobacterium and Streptomyces. Species of the genus Microbacterium have been reported to be endophytes of marigold (Calendula officinalis), rice, common bean (Phaseolus vulgaris), potato (Solanum tuberosum) and maize (Zinniel et al. 2002; Sturz and Kimpinski 2004). In addition, the Streptomyces strain EN27 has been reported to be an endophyte of wheat (Triticum aestivum) seeds (Coombs and Franco 2003). Interestingly, both Streptomyces isolates identified in our study (UYFA153 and UYFA156) were obtained from surface-desinfected tall fescue seeds. On the other hand, Curtobacterium spp. have been reported to be endophytes of red clover (Trifolium pratense), potato, strawberry (Fragaria spp.) and Eucalyptus spp. (Sturz et al. 1997; Zinniel et al. 2002; Procópio et al. 2009; de Melo Pereira et al. 2012).

Finally, for Firmicutes, isolates were identified as Bacillus, and they also came from surface-desinfected seeds of tall fescue. Bacillus isolates have been reported to be associated with and/or endophytic within several important crops, including citrus species, maize, cotton (Gossypium spp.) and carrots (Daucus carota) (McInroy and Kloepper 1994; Araújo et al. 2001; Surette et al. 2003). In particular, the partner B. pumilus JP12 strain–tall fescue has been demonstrated to be very successful in the phytoremediation of soils contaminated with heavy metals (Lu et al. 2013; Lu and Zhang 2014).

To our knowledge this study reveals several novel isolates associated with tall fescue, belonging to the genera Bacillus, Streptomyces, Microbacterium, Xanthomonas, Acidovorax, Variovorax, Pantoea and Acinetobacter. These isolates possess a number of PGP traits and consequently are excellent candidates as PGP inoculants in biotechnological application.

Isolates associated with tall fescue can promote its growth

Isolates selected according to their in vitro PGP traits were evaluated as inoculants under gnotobiotic conditions in two tall fescue cultivars, cv. SFRO Don Tomás and the commercial cv. Tacuabé. Three sets of PGP isolates were identified in cv. SFRO Don Tomás under the conditions studied. The first set (UYFA09, UYFA15, UYFA22, UYFA157, UYFA249) was able to promote the height of stems, the second set (UYFA68) promoted the dry weight of the plants and the third set (UYFA61, UYFA156, UYFA215) was able to promote both the aforementioned parameters. It is interesting to note that all of the isolates identified as belonging to the genus Microbacterium were able to promote plant growth. Microbacterium strains have been reported as growth promoters of apple trees (Malus domestica) and potato (Sessitsch et al. 2004; Karlidag et al. 2007), and the authors of both of these studies speculated that the ability to fix N₂ and to produce IAA were most probably the mechanisms involved in the reported PGP effects. In the present study, Microbacterium isolates UYFA26, UYFA61 and UYFA68 were defined as IAA producers, but they were negative for all of the other PGP traits tested, while isolate UYFA09 was nifH positive and negative for all others PGP traits. Therefore, it is reasonable to speculate that these PGP traits are also involved in the PGP effects observed with these isolates.

The seed-borne Streptomyces isolate UYFA156 was able to increase both plant dry weight and stem height. Streptomyces strains are well known for their ability to produce a wide range of secondary metabolites (Castillo et al. 2002; Nassar et al. 2003). In addition, several Streptomyces isolates have been described as growth promoters of many crops, such as chickpea (Cicer arietinum), wheat, soybean (Glycine max), sorghum and rice, through both indirect (biocontrol) and/or direct mechanisms (phytostimulation) (Nassar et al. 2003; Gopalakrishnan et al. 2011; Sadeghi et al. 2012). In these studies, the isolates possessed several PGP traits, such as IAA production, Fe and P solubilization and cellulase, protease, lipase and chitinase activites. Moreover, the Streptomyces isolate, S. griseoluteus, was found to be capable of producing relatively high levels of polyamines, which were involved in the observed PGP effect (Nassar et al. 2003). Our data show that isolate Streptomyces sp. UYFA156 is a diazotroph which tested negative for the other PGP traits examined. Therefore, it can be speculated that this mechanism may be involved in the PGP effect observed in this strain in cv. SFRO Don Tomás.

Curtobacterium sp. UYFA157 was also able to promote both growth parameters evaluated. Curtobcterium spp. have been reported as plant growth promoters of cucumber and other crop plants through the biological control of plant diseases (Raupach and Kloepper 1998; Ramamoorthy et al. 2001). Nevertheless, we classified isolate UYFA157 as a diazotroph; therefore, it is possible that this mechanism is involved in the observed PGP effect.

Regarding the PGP isolates Pantoea sp. UYFA15 and Pseudomonas sp. UYFA249, the former was described as a diazotroph, while the latter was able to produce IAA and siderophores. Pantoea spp. have been reported to be PGPB of several important agronomical crops, such as maize, rice, sugarcane, sweet sorghum, canola, lentil (Lens culinaris) and pea (Pisum sativum) (Verma et al. 2001; Sergeeva et al. 2007; Montañez et al. 2012; Taulé et al. 2012; Quecine et al. 2012; Mareque et al. 2015). Pseudomonas spp. are well-known PGPB of several plants, such as olive tree (Olea europaea), spruce (Picea), Eucalyptus, cactus (Pachycereus pringlei), tomato (Solanum lycopersicum) and wheat (Chanway et al. 2000; Zhang et al. 2000; Ran et al. 2005; Shaharoona et al. 2007; Puente et al. 2009; Richardson et al. 2009; Mercado-Blanco and Lugtenberg 2014). In some of these studies the mechanism involved in the PGP effects included P and N uptake, growth stimulation and IAA production (Richardson et al. 2009). Therefore, it is possible that the production of IAA and siderophores by isolate UYFA249 is involved in the PGP effect observed in tall fescue.

Finally, the nifH-positive isolate UYFA22, which has 100 % identity with Variovorax paradoxus strain S110, was able to promote the stem height of tall fescue cv. SFRO Don Tomás. Variovorax spp. have been described as PGPB of pea and maize in which the probable PGP mechanism involved is ACC-deaminase activity (Dodd et al. 2009).

In contrast to cv. SFRO Don Tomás, our PGP assays showed that only the two isolates related to Rhanella, UYFA289 and UYFA337, were able to promote the growth of the commercial tall fescue cv. Tacuabé. Both isolates are described here as diazotrophs, while UYFA288 was also able to produce IAA. Both isolates were closely related to the type strain of Rahnella aquatilis AJ233426. R. aquatilis strains have been reported to be PGPB of several important agronomical crops, such as apple, tomato, barley (Hordeum vulgare), chickpea, pea and maize (Calvo et al. 2007; Vyas et al. 2010).

It is interesting to note that the ability of different isolates to promote plant growth was dependent on the cultivar. Moreover, a larger number of PGPB isolates were observed for cv. Don Tomás, highlighting the fact that plant-associated bacteria are often plant genotype dependent since the bacterial collection was constructed from cv. Don Tomás (Long et al. 2008).

In all of the PGPB strains mentioned here, additional studies are required to determine which mechanism is involved in their PGP abilities. However, regardless of the mechanism put into play in the growth promotion test under gnotobiotic conditions, the significance of this work lies in the diversity of bacteria with proven PGP capacity which were identified under in vitro and in vivo conditions. To our knowledge, this is the first report in which isolates belonging to the genera Microbacterium, Pantoea, Variovorax, Streptomyces and Pseudomonas are reported as PGPB in tall fescue.

From the biotechnology perspective, PGP experiments will be performed with this set of isolates as inoculants under greenhouse conditions. Special attention will be paid to isolates UYFA61, UYFA156 and UYFA215, which we found promoted both stem height and plant dry weight.

Concluding remarks

The results of our study reveal that a wide variety of putative endophytes are associated with the seeds, roots and stems of tall fescue cv. SFRO Don Tomás and that these also harbor several PGP and plant infection features. Ten of the isolates were able to promote the growth of cv. SFRO Don Tomás under gnotobiotic conditions, of which four were able to promote both growth parameters evaluated (stem height and plant dry weight). In addition to reporting known phenotypes, we describe novel strains for the first time as putative endophytes associated with tall fescue as well as PGPB for this crop. These results stress the biotechnology potential of the bacterial collection constructed in this study, and the PGPB strains identified will be tested in a more complex system with the aim of producing a PGP inoculant for tall fescue cv. SFRO Don Tomás.