Isolation, characterization and plant growth promotion effects of putative bacterial endophytes associated with sweet sorghum (Sorghum bicolor (L) Moench)

Abstract

Sweet sorghum (Sorghum bicolor) is cultivated in Uruguay in complementation with sugarcane (Saccharum officinarum) as a feedstock for bioethanol production. It requires the application of high levels of chemical fertilizer for optimal growth, which causes environmental degradation. Plant growth-promoting (PGP) bacteria are of biotechnological interest since they can improve the growth of several important agronomical crops. Of particular interest are endophytes, which are those bacteria that can be detected at a particular moment within the internal tissues of healthy plants from where they can promote their growth. The aims of this work were to isolate and characterize, as well as identify putatively endophytic bacteria associated with sweet sorghum (cv-M81E), and also to study the inoculation effects of selected isolates on sorghum growth. A collection of 188 putative endophytes from surface-sterilized stems and roots was constructed and characterized. Bacterial isolates were shown to belong to different genera including Pantoea, Enterobacter, Pseudomonas, Acinetobacter, Stenotrophomonas, Ralstonia, Herbaspirillum, Achromobacter, Rhizobium, Chryseobacterium, Kocuria, Brevibacillus, Paenibacillus, Bacillus and Staphylococcus. PGP and infection features were investigated in vitro, and revealed some promising biotechnological candidates. In addition, isolates UYSB13 and UYSB45 showed PGP effects in greenhouse assays. This work provides the basis for further studies under field conditions, with the final aim of developing an effective inoculant for sorghum.

Introduction

Fossil energy resources are depleting dramatically in order to meet the increasing world energy demands. Moreover, climate change caused by carbon emissions from fossil fuels reinforces the need to search for alternatively energy sources. Crop plants are one of the best sources of renewable energy, as they can be used as feedstock for biofuel production. With this aim, several complementary crops are cultivated in Uruguay, such as sugarcane (Saccharum officinarum) and sweet sorghum (Sorghum bicolor (L.) Moench) (Kim and Day 2011; Ratnavathi et al. 2011). Globally, sweet sorghum is the fourth most important cereal, and is known as a multipurpose crop since it is used for grain, forage, syrup, fodder and bioethanol production (Almodares and Hadi 2009; Shoemaker and Bransby 2010). Nevertheless, this crop has a high demand for chemical fertilizer for optimal productivity, resulting in leaching and run-off of nutrients, especially nitrogen (N) and phosphorus (P), leading to environmental degradation (Adesemoye and Kloepper 2009). These problems emphasize the need for new technologies in agriculture with the aim of attaining more sustainable production systems. A promising alternative to chemical fertilization is the use of plant growth-promoting bacteria (PGPB) (Lugtenberg and Kamilova 2009). Amongst these, bacterial endophytes are referred to as those bacteria that can be detected at a particular moment within the internal tissues of an apparently healthy host plant (Hallmann et al. 1997; Schulz et al. 2006). In contrast to phytopathogenic bacteria, they do not cause any disease symptoms; indeed, they can promote plant growth (James 2000; Berg 2009). Mechanisms involved in endophytic plant growth promotion (PGP) could be direct or indirect. Direct PGP mechanisms include biological nitrogen fixation (BNF) and mineral solubilisation (P, Fe), as well as the production of plant phytohormones (auxins, cytokinins and gibberellins), while indirect mechanisms include biocontrol against phytopathogens mediated by antibiotics, competition for nutrients and niches, or the induction of an induced systemic resistance (ISR) response (Rosenblueth and Martínez-Romero 2006; Mei and Flinn 2010; Compant et al. 2010). A number of reports have demonstrated the association of sweet sorghum with several bacterial endophytes belonging to the genera Herbaspirillum, Azospirillum, Klebsiella, Enterobacter, Burkholderia, Paenibacillus and others (Olivares et al. 1996; Budi et al. 1999; Zinniel et al. 2002; Grönemeyer et al. 2011). Moreover, a PGP effect was demonstrated for several diazotrophic endophytes, such as Azospirillum lipoferum, A. amazonense, Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus, when they were inoculated onto sweet sorghum under greenhouse and field conditions (Pereira et al. 1988; Sarig et al. 1990; Chiarini et al. 1998). In consideration of this, the management of the interaction between endophytes and their hosts (such as sweet sorghum) might play a significant role in the development of more sustainable agricultural production systems. In Uruguay, the major sweet sorghum cultivar used by the producers is cv. M81E, which is of national interest. However, until now, no studies have been conducted in Uruguay on the native microorganisms naturally associated with sweet sorghum or have evaluated their potential PGP capability. The aims of this work were: (1) to obtain a collection of culturable putatively endophytic bacteria associated with sweet sorghum cv. M81E, (2) to characterize the collection based on PGP and infection features and thus to identify isolates of interest, and (3) to study the inoculation effects of selected isolates on sweet sorghum growth. The data obtained will contribute to future research aimed at developing sweet sorghum inoculants based on native PGPB specifically for cv. M81E.

Materials and methods

Isolation of putatively endophytic bacteria associated with sweet sorghum

With the aim of isolating putative endophytes associated with the commercial sweet sorghum cv. M81E, two approaches were employed. In the first one, bacteria were isolated from the roots and stems of trap plants. For this, sterilized seeds were sowed into pots containing sterile sand and soil from the sweet sorghum cropping region, Bella Union, Artigas, Uruguay (30°37′56′S, 57°21′18′W, 120 m asl). In the second approach, bacteria were isolated from seeds, roots and stems of plants collected directly from the field in the same cropping region. The same surface-sterilization protocol was used for seed and plant material in both cases. Briefly, 10 g of material were incubated for 5 min in 70 % EtOH, then 20 min in 4 % sodium hypochlorite, and finally rinsed 4 times with sterile deionized water. Sterilized seeds for trap plants were germinated on 0.8 % water agar plates before sowing. On the other hand, for bacterial isolation from roots, stem and seeds, they were aseptically macerated in a solution of 0.9 % NaCl supplemented with cyclohexamide (100 μg μl⁻¹). Serial dilutions from the suspensions obtained, were inoculated onto agar plates containing DYGs and LGI media in the case of trap plants, or TSA (Tryptic Soy Agar; Difco) medium in the case of field plants. Those culture media were selected with the aim to have a high diversity of heterotrophic bacteria isolates. All isolates obtained were purified and stored at −80 °C in 50 % glycerol.

Screening for biofertilization activities

With the aim of detecting putatively diazotrophic isolates, the whole collection was subjected to nifH PCR amplification by using the primers PolF (5′-TGCGAYCCSAARGCBGACTC-3′) and PolR (5′-ATSGCCATCATYTCRCCGGA-3′) (Poly et al. 2001). In all cases, cell lysates were used as templates (Rivas et al. 2001) and a single colony resuspended in water as a starting material. The PCR mixture was 2.5 μl 10× Fermentas Taq reaction buffer, 3.0 mM MgCl₂, 0.16 mM dNTP’s, 0.8 μM of both set of primers, 0.5 U Fermentas Taq polymerase, 4 % BSA and 4.0 μl of a cell lysate template, in a final reaction volume of 25 μl. The PCR conditions were as follows: 1 cycle at 95 °C for 5 min; 30 cycles at 95 °C for 45 s; 58 °C for 45 s, and 72 °C for 30 s; and a final cycle at 72 °C for 5 min. The amplification products were analyzed by 1 % (w/v) agarose gel electrophoresis in TAE buffer and stained with GoodView (Beijing SBS; Genetech).

In addition, the ability to fix N₂ was tested in those isolates, which harbored the nifH gene, in vials containing LGI, LGI-P and JNFb N-free semisolid media (Reis et al. 1994; Perin et al. 2006). The vials were incubated at 30 °C for up to 7 days and those which showed a growth pellicle were replicated into a new fresh vial containing the same media with the aim of confirming the presence of the growth pellicle (Baldani et al. 2014).

Inorganic phosphate solubilization ability and siderophore production were tested on plates containing either GL rich (Sylvester-Bradley et al. 1982) or chromeazurol medium (Schwyn and Neilands 1987), respectively. After 72 h of growth, the presence of a translucent or yellow halo around the colony was evaluated, indicating phosphate solubilizing or siderophore-producing isolates, respectively. In the case of the siderophore production assay, Herbaspirillum seropedicae Z67 was used as a positive control (Rosconi et al. 2013).

Production of enzymes for phytostimulation

An assay for the quantitative detection of indole-3-acetic acid (IAA) production was performed on all the bacterial collection by using a colorimetric method (Sarwar and Kremer 1995) as described previously (Taulé et al. 2012).

With the aim of detecting aminocyclopropane-1-carboxylate (ACC) deaminase activity, all the isolates were grown overnight at 28 °C in a 96-microwell plate containing 2 ml of LGI+N medium to mid- to late-log phase. Cultures were harvested by centrifugation at 4,000 g for 20 min, the supernatants were removed and the pellets were washed twice with 1 ml of LGI−N medium. The washed cells were resuspended in 150 μl of LGI N-free medium and an aliquot of 5 μl was inoculated onto plates containing solid LGI N-free plates complemented with 5 % N and 30 mmol plate⁻¹ of ACC (Penrose and Glick 2003). All plates were supplemented with 1.8 % Bacto-Agar (Difco). Plates were incubated for 3 days at 30 °C. Isolates, which were able to growth with ACC as a sole source of N, were considered as positive.

Screening for hydrolytic activity

For determination of hemicellulose, cellulose and peroxidases activities, the bacterial collection was grown in plates containing TSA solid medium supplemented with 0.5 % Avicel (Sigma-Aldrich, Germany) 0.2 % cellulose or 250 mg l⁻¹ ABTS (Sigma-Aldrich) with and without the addition of 0.1 g l⁻¹ MnCl₂·4H₂O, respectively. In the case of protease activity detection, plates containing TSA medium were supplemented with 5 % (w/v) of skimmed milk (Hofrichter and Fritsche 1997; Kim et al. 2008; Martinez-Rosales and Castro-Sowinsky 2011).

Screening for biofilm formation

Biofilm formation was detected using the crystal violet (CV) method (Christensen et al. 1985). For this, the collection was first grown at 30 °C with agitation, in 96-microwell plates containing 200 μl of TSB (Tryptic Soy Broth; Difco) medium, until an optical density at 620 nm of 0.2 (DO_620nm = 0.2). The plates were then incubated for 48 h at 30 °C without agitation, the supernatant was removed, and the plates were washed with 1× phosphate buffered saline (PBS). For staining, a 0.1 % CV solution was added (200 μl per well) and the plates were incubated for 20 min. The excess CV was removed by washing the plates under running tap water while the bound CV was released from the cells by adding 200 μl of 95 % EtOH. The absorbance of the suspension was measured at 570 nm. All steps were carried out at room temperature (Peeters et al. 2008).

Evaluation of bacterial genomic diversity by ERIC-PCR

With the aim of evaluating the genomic diversity of the selected isolates associated with sweet sorghum cv. M81E, the Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) technique was employed as described previously (Taulé et al. 2012). Data were further analyzed using GelComparII6.5 (Applied Maths) software. The similarity between strains was expressed by Dice’s coefficient, and cluster analysis was performed using the unweighted pair group average linkage method (UPGMA) with a similarity of 80 %.

16S rRNA gene amplification, sequencing and phylogenetic analysis

Amplification and sequencing of the 16S rRNA gene was performed for selected isolates as described before (Taulé et al. 2012). The 16S rRNA gene sequences were deposited in GenBank with the following accession numbers: KJ532081–KJ532123.

The quality of the sequences obtained was checked manually and assembled using DNA Baser Sequence Assembler v.3.× (2010) (http://www.dnabaser.com). Sequences were imported into the ARB software package v.5.5 (Ludwig et al. 2004) and added to the database. Sequences were aligned using ARB FastAligner, then refined manually. Phylogenetic trees were generated using the maximum parsimony, neighbor joining and maximum likelihood algorithms with 1,000 bootstrap replicates.

Capacity of the isolates to grow using different C, N sources and with various antibiotics

The entire bacterial collection was grown on plates containing LGI medium supplemented with the C or N sources to be tested at the final concentrations as the original protocol (Cavalcante and Dobereiner 1998). The C-sources analyzed were maltose, mannitol, glucose, sucrose, malate, fructose, lactose, sucrose, glycerine, pyruvic acid and vinasse (byproduct generated during the distillation of ethanol, yeast, amino acids and/or organic acids from cane molasses fermentation mixtures); the last two were obtained from industrial waste. In the case of the N-sources tested, these were (NH₄)₂SO₄, KNO₃, NH₄Cl, L-tyrosine, L-asparagine and L-glutamic acid as well as urea.

For antibiotic resistance determination, TSA plates were supplemented with gentamicin, kanamycin, streptomycin, spectinomycin, ampicillin, polymixina b, tetracycline or nalidixic acid, all at a final concentration of 100 μl ml⁻¹, as well as neomycin at a final concentration of 25 μl ml⁻¹.

Plant growth promotion of sweet sorghum inoculated with selected isolates

The growth response of sweet sorghum to bacterial inoculation was studied in greenhouse conditions. Seeds were surface sterilized as described above and incubated for 45 min with slow agitation with a suspension of 1.0 x10⁸ cells ml⁻¹ of each isolate to be tested. Inoculated seeds were germinated on 0.8 % water agar for 2 days, transferred to pots containing 1.5 kg of sand:soil (1:2) as substrate and maintained in greenhouse conditions with a photoperiod of 8/16 h light/dark. After 30 days post-inoculation (pi), plants were re-inoculated. This experiment had 5 treatments with 10 replicates in a completely randomized design. The isolates tested as inoculants were Rhizobium sp. UYSB12, Bacillus sp. UYSB13, Enterobacter sp. UYSB34 and Pantoea sp. UYSB45. As a negative control, a treatment containing plants without inoculation was employed. Additionally, plants inoculated with the known PGPB strain Azospirillum brasilense Sp7 were used as a positive control reference treatment. At 3 months pi, the experiment was harvested, roots and aerial parts were dried at 60 °C until constant weight, and their dry weights then determined.

Statistical analysis

Statistic analyses were done with the Infostat programme, and in those treatments where significant differences were confirmed the Fisher LSD test (p < 0.10) was employed (InfoStat 2008).

Results

Isolation of putatively endophytic diazotrophic bacteria associated with sweet sorghum cv. M81E

A library containing 188 isolates from surface sterilized sweet sorghum seeds (2 isolates), stems (79 isolates) and roots (107 isolates) was constructed. As they were isolated from surface-sterilized material, they can be considered to be putative endophytes until proven by microscopy. The plant material was collected from a crop region in which plants were grown with low level of N fertilization. With the aim of isolating a broad range of bacteria, various isolation approaches (from trap plants and field material), as well as different bacterial growth media (DYGs, LGI and TSA) were employed. With regard to the growth media, 62, 44 and 82 isolates were isolated from DYGs, LGI and TSA media, respectively. The genomic diversity of the entire bacterial collection was analyzed by ERIC-PCR, and 6 different groups with a similarity value of 80 % were found using the GelComparII6.5 software (Table 1). In addition, 58 putatively diazotrophic isolates were detected in the collection by PCR amplification of the nifH gene (Table 1). These latter isolates were tested for their availability to grow in LGI, LGI-P and JNFb semi-solid N-free media. Using this approach, 38 isolates capable of producing a growth pellicle were distinguished (Table 1).

Table 1 Plant growth promotion features of putatively endophytic bacterial isolates from sweet sorghum cultivar M81E

Characterization of sweet sorghum associated isolates

With the aim of detecting plant growth promotion features, the whole collection was screened for the ability to produce IAA, siderophores, solubilise phosphates and the presence of ACC-deaminase activity. Out of 188 isolates tested, 33 and 18 were able to produce IAA and siderophores, respectively, 22 to solubilize phosphate, while 130 presented ACC activity (Table 1). In addition, the collection was also screened for the presence of plant infection traits, including hemicelluloses, cellulose, protease, peroxidase activities and biofilm formation. Results showed that only 3 and 5 isolates presented hemicellulase and cellulase activity, respectively, while 26 and 41 isolates presented protease activity and biofilm formation ability, respectively (Table 1).

Physiological features of the bacterial isolates

The ability to grow in different C and N sources, as well as in the presence of antibiotics in the culture media, was evaluated for the entire bacterial collection. As sole C-source, 97 % of the isolates were able to grow in fructose or crystal sugar, 94 % in glycerine, 92 % in lactose or glucose, 86 % in malic acid, 82 % in mannitol, 70 % in pyruvic acid, 38 % in EtOH, and 26 % in maltose (Table S2). As sole N-source, 99 % of the isolates were able to grow in L-asparagine, 94 % in KNO₃, 93 % in L-tyrosine, 89 % in urea, 88 % in L-glutamic acid, 63 % in (NH₄)₂SO₄ and none were able to grow in the presence of NH₄Cl (Table S2).

When the capacity to grow in the presence of various antibiotics was evaluated, it was found that 100% of the isolates were able to grow in the presence of neomycin, 98 % in ampicillin, 96 % in spectinomycin, 89 % in streptomycin, 81 % tetracycline, 77 % in gentamycin, 66 % in polymyxin B, 63 % in nalidixic acid and 43 % in kanamycin (Table S2).

Identification and phylogenetic analysis of sweet sorghum associated isolates based on their partial 16S rRNA sequences

For 16S rRNA gene sequencing and analysis, isolates were selected according to their in vitro PGP features and their ERIC genomic patterns. As shown in Table S1, BLAST searches against the NCBI database revealed close relationships to known plant-associated bacteria, including genera belonging to the phyla Alphaproteobacteria (Rhizobium), Betaproteobacteria (Achromobacter, Herbaspirillum and Ralstonia), Gammaproteobacteria (Acinetobacter, Enterobacter, Pantoea, Pseudomonas, Serratia and Stenotrophomonas), Actinobacteria (Kocuria), Firmicutes (Brevibacillus, Bacillus, Paenibacillus, and Staphylococcus) and Bacterioidetes (Chryseobacterium).

A phylogenetical tree (dendrogram) was constructed based upon the 16S rRNA gene sequences of 34 selected isolates (Fig. 1). Since the largest number of isolates associated with sweet sorghum belonged to the Gammaproteobacteria, this group will be described first. As can be seen from Fig. 1, Pantoea isolates UYSB38, UYSB39, UYSB43, and UYSB45 grouped in a cluster with two branches, within which isolates UYSB39 and UYSB45 clustered closely with the reference strains P. wallisii and P. dispersa, respectively, but none of the other isolates clustered close to any reference strains. The 16S rRNA sequences of isolates UYSB05, UYSB22 and UYSB34 showed that they belonged to the genus Enterobacter (Fig. 1, Table S1) and that they grouped in one well supported cluster distant from any reference strain. In addition, isolate UYSB48, which has an identity of 99 % with Serratia marcescens WW4 (Table S1), shared the same node as Enterobacter spp., but was located in a different branch clustered closely with the reference species Serratia marcescens subsp. marcescens.

Fig. 1

Neighbor-joining phylogenetic tree based on bacterial 16S rRNA sequences of representative isolates. The tree shows the phylogenetic affiliation of 34 partial 16S rRNA sequences of endophytic bacterial isolated from sweet sorghum (Sorghum bicolor). Numbers at branches represent bootstrap values >50 % from 1,000 replicates. Thermaerobacter litoralis was used as an outgroup. The scale bar shows the number of nucleotide substitutions per site

Pseudomonas and Acinetobacter spp. shared the same node, and clustered in two separated branches. With regard to the Acinetobacter spp., isolate UYSB41 clustered closely related with the reference strain A. parvus, while isolate UYSB42 grouped in a different branch distant from any reference strain. The only isolate related to the genus Pseudomonas in the tree, UYSB37 (Table S1; Fig. 1), clustered in a branch distant from any reference strain.

Finally, from the Gammaproteobacteria phylum, isolates UYSB32 and UYSB33, which belong to the genus Stenotrophomonas (Table S1), were located in a branch totally separate from the other Gammaproteobacteria isolates, and grouped together in a cluster closely related to the reference species Stenotrophomonas maltophilia.

Betaproteobacteria spp. were also present in the collection and they grouped in a well supported cluster with three different branches (Fig. 1). Isolate UYSB09, which belonged to the genus Achromobacter (Fig. 1; Table S1), did not group with any reference strain, but shared the same node as the reference strains A. xylosoxidans, and A. marplatensis. Additionally, isolates UYSB01 and UYSB02, which on the basis of their 16S rRNA gene sequences were similar to Ralstonia spp. (Table S1), grouped together in a cluster distant from any reference strain (Fig. 1). Finally, from the Betaproteobacteria, the only isolate represented in the genus Herbaspirillum UYSB35, although it clustered together with Herbaspirillum spp., it was in a separate branch distant from any reference strain (Fig. 1).

With regard to the Alphaproteobacteria (Fig. 1), the 16S rRNA sequences of isolates UYSB11, UYSB12, UYSB13, and UYSB25 indicated that they belonged to the genus Rhizobium (Table S1). From this group, isolates UYSB12, UYSB13 and UYSB25 grouped closely with the reference strains R. pusense, R. skierniewicense and R. mesosinicum, respectively, while isolate UYSB11 grouped alone distant from any reference strain.

The only isolate in the Bacterioidetes phylum, UYSB46, grouped closely with the reference species Chryseobacterium formosense (Fig. 1).

The Firmicutes was well represented in the collection (Table S1), and the dendogram based on 16S rRNA sequences showed that isolates from this class grouped in a single cluster (Fig. 1). The genus Bacillus was represented in the collection by the isolates UYSB06, UYSB07, UYSB15, UYSB18, UYSB20, and UYSB26 (Table S1). Isolates UYSB07, UYSB15, UYSB20, and UYSB26 grouped closely related with the reference species B. acidiceler, B. pumilus, B. niacini and B. megaterium, respectively, while the remaining isolates clustered distant from any reference strain. In addition, the only isolate representing the genus Staphylococcus UYSB03, shared the same node as the Bacillus isolate UYSB15, and grouped closely with the reference strain Staphylococcus epidermidis.

With regard to the genus Brevibacillus, isolates UYSB29 and UYSB36 (Table S1) grouped in a single well-supported cluster (Fig.1), while isolate UYSB29 did not cluster with any reference strain, isolate UYSB36 was closely related to the reference species B. laterosporus.

Finally, from the Firmicutes, isolates UYSB19 and UYSB27, which belonged to the genus Paenibacillus (Table S1), were grouped in a well-supported cluster (Fig.1); isolate UYSB27 grouped closely with the reference species P. glycanilyticus while isolate UYSB19 grouped in a separate branch distant from any reference strain.

Finally, and with regard to the only isolate from the Actinobacteria (Table S1), the dendrogram based on 16S rRNA sequences shows that isolate UYSB08 grouped closely with the reference species Kocuria palustris.

Plant growth promotion of sweet sorghum inoculated with selected bacterial isolates under greenhouse conditions

Isolates tested as inoculants on sweet sorghum were selected from the bacterial collection according to their in vitro PGP features and their phylogenetic affiliations. Inoculated plantlets were grown under greenhouse conditions in pots containing sterile sand and soil as a substrate. Four-month pi plants were harvested and biometric parameters as well as total N content (%) were measured (Table 2). None of the isolates showed any significant differences from the control when stem diameter was evaluated, but isolates Rhizobium sp. UYSB13 and Pantoea sp. UYSB45 showed significant differences from the negative control in their stem height and dry weight (roots and shoots) (Table 2). Additionally, isolates Rhizobium sp. UYSB12 and Enterobacter sp. UYSB34 showed significant differences from the control only in their root and the shoot dry weights, respectively. Finally, A. brasilense Sp7, which was used as a reference strain, did not show any significant differences from the negative control in all the parameters evaluated.

Table 2 Effects of inoculation with putative bacterial endophytes on the growth of sweet sorghum cv. M81E

Discussion

A collection containing 188 isolates associated with roots and stems of the commercial sweet sorghum cv. M81E was constructed and characterized. Out of these, 57 isolates were nifH ⁺ from which 52 were able to produce a growth pellicle in semi-solid N-free media, and so they were defined as diazotrophs. In addition, a number of isolates with in vitro biofertilization, phytostimulation and infection features were also detected in the collection, suggesting their biotechnological potential as inoculants for this cultivar. Phylogenetic analysis revealed a high diversity of bacteria associated with sweet sorghum including the genus Rhizobium from the Alphaproteobacteria, the genera Achromobacter, Herbaspirillum and Ralstonia from the Betaproteobacteria, and a high number of isolates related to the Gammaproteobacteria, including the genera Pseudomonas, Acinetobacter, Pantoea, Enterobacter and Stenotrophomonas. In addition, an isolate belong to the Bacterioidetes was detected, sharing 98 % 16S rRNA sequence identity with the reference strain Chryseobacterium formosense CC-H3-2. Interestingly, Gram-positive isolates were also detected, including one isolate belong to the Actinobacteria, which was related to the genus Kocuria, and 13 isolates belonging to the Firmicutes, which were related to the genera Brevibacillus, Paenibacillus, Staphylococcus and Bacillus.

In this study, the plants used for bacterial isolation were healthy, but some of the genera identified have been reported as phytopathogens, such as the plant-associated R. radiobacter (formerly Agrobacterium tumefaciens), which is the causal agent of crown gall. Moreover, several isolates were of clinical importance, such as Stenotrophomonas maltophila, Pseudomonas putida, Staphylococcus epidermidis and Ralstonia spp. Nevertheless, most of the genera identified have been reported as being associated with a number of important agronomical crops including sweet sorghum (Hallmann et al. 2006; Rosenblueth and Martínez-Romero 2006). The most abundant and diverse phylum of bacteria in the collection was the Gammaproteobacteria. Interestingly, the same results were obtained from a collection of putative endophytes associated with sugarcane varieties cultivated in Uruguay (Taulé et al. 2012). Within the Gammaproteobacteria, isolates of the genera Enterobacter, Pantoea (Enterobacteriales), Pseudomonas, Acinetobacter (Pseudomonales), and Stenotrophomonas (Xanthomonadales) have been previously reported as being endophytes and/or associated with different Poaceaous crops (Hallmann et al. 2006; Rosenblueth and Martínez-Romero 2006; Beneduzi et al. 2013). In particular, Enterobacter and Pseudomonas isolates were reported as associated with sweet sorghum plants from the state of Nebraska in the USA (Zinniel et al. 2002), but no isolates belonging to the Gammaproteobacteria were described in a collection of bacteria associated with sweet sorghum from the Kavango region of Namibia (Grönemeyer et al. 2011). To our knowledge, this is the first report in which isolates of the genera Pantoea, Acinetobacter and Stenotrophomonas were described as being associated with sweet sorghum.

The Alphaproteobacteria was represented in this study by strains of the genus Rhizobium. The phylogenetic analysis revealed two different groups within this genus: one closely related to the phytopathogen R. radiobacter and the non-nodulated R. pusense reference strains, while the other group was more closely related to plant-associated or nodulating Rhizobium species including R. mesosinicum, R. alamii and R. sullae. There have been several reports of the isolation of rhizobia as endophytes and/or associated with various non-legume crops (Hallmann et al. 2006; Rosenblueth and Martínez-Romero 2006; Taulé et al. 2012; Beneduzi et al. 2013), but until the present study none came from sweet sorghum. The genera Ralstonia, Achromobacter, and Herbaspirillum of the Betaproteobacteria were also present in our collection. Some species of the genus Ralstonia have been reported to be associated with roots of sweet sorghum from Namibia as well as being endophytes of soybean (Kuklinsky-Sobral et al. 2005; Grönemeyer et al. 2011). Species of the genus Achromobacter were previously described as putative endophytes associated with Citrus spp., sunflower (Helianthus annuus) and sugarcane, as well as rhizobacteria associated with canola (Brassica napus) (Araújo et al. 2001; Bertrand et al. 2001; Ambrosini et al. 2012; Taulé et al. 2012). In addition Herbaspirillum spp. have been isolated and reported as associated (including endophytically) with various crops, such as rice (Oryza sativa), maize (Zea mays), sugarcane and sweet sorghum (Olivares et al. 1996; Monteiro et al. 2012), but to our knowledge there are no previous reports of Achromobacter and Rhizobium species associated with sweet sorghum plants.

The Bacterioidetes phylum was represented by only one isolate, which belongs to the genus Chryseobacterium. Species of this genus were described as endophytes of several plants, such as cucumber (Cucumis sativus), potato (Solanum tuberosum), canola and tomato (Solanum lycopersicum) (Hallmann and Berg 2006), but there are no previous reports of them being associated with sweet sorghum.

With regarding to the Gram-positives, there were isolates related to the Actinobacteria (Kocuria spp.) and Firmicutes (Paenibacillus, Brevibacillus, Staphylococcus and Bacillus spp.). Strains from the genus Bacillus are well reported as endophytes and/or as associated bacteria to several crops (Hallmann and Berg 2006; Rosenblueth and Martínez-Romero 2006). In particular, Bacillus spp. were reported as isolated from the rhizosphere of sorghum plants and described as biocontrol agents, but not as endophytes or PGPB of this crop (Budi et al. 1999; Martinez-Absalon et al. 2012). In the case of Paenibacillus spp., isolates were described as endophytes of cucumber, sweet potato (Ipomoea batatas), and sweet sorghum (Hallmann and Berg 2006; Rosenblueth and Martínez-Romero 2006; Grönemeyer et al. 2011). Regarding the genus Staphylococcus, numerous isolates were reported as endophytes associated with various plants, such as carrots (Daucus carota), sugarcane and Alyssum bertolonii (Surette et al. 2003; Barzanti et al. 2007; Velázquez et al. 2008). Additionally, Brevibacillus spp. have also been reported as endophytes associated with maize and cotton wood (Populus deltoides), as well as in balloon flower plants (Platycodon grandiflorum) (Asraful Islam et al. 2010; Grönemeyer et al. 2011; Brown et al. 2012), but there are no previous reports of strains from these genera associated with sweet sorghum.

Finally, from the Actinobacteria, the literature shows that Kocuria spp. were isolated from marigolds (Tagetes spp.) and reported as biocontrol agents (Sturz and Kimpinski 2004), but to our knowledge this is the first report in which an isolate from this genus was reported as a putative endophyte associated with sweet sorghum.

Thus, our study reveals several novel isolates associated with sweet sorghum with plant growth-promoting characteristics, and which are, therefore, excellent potential candidates for biotechnological application as a PGP inoculant.

Plant growth promotion of putative endophytes associated with sweet sorghum

Isolates tested as an inoculant on cv. M81E of sweet sorghum were selected from the collection taking into consideration their PGP features as well as their 16S rRNA-based identity. The results demonstrated that isolates Rhizobium sp. UYSB13 and Pantoea sp. UYSB45 were PGPB under the conditions tested (Table 2). In the present study, Azospirillum brasilense Sp7, which is well reported as a PGP of sorghum (Steenhoudt and Vanderleyden 2000; Lucy et al. 2004; Bashan and Luz 2010), was also tested as inoculant in the PGP assays, but no significant PGP effects were obtained for this strain in any of the parameters measured under the studied conditions (Table 2). This absence of a PGP effect by A. brasilense Sp7 could be due to plant genotype specificity or to poor competition with the microbiota present in the soil used in the PGP assays (Long et al. 2008).

Rhizobia, such as Rhizobium leguminosarum, Bradyrhizobium japonicum and Sinorhizobium meliloti, are often reported as PGPB in non-legume plants, such as maize, rice and sweet sorghum (Rashad et al. 2001; Matiru and Dakora 2004; Bhattacharjee et al. 2008). In this study, Rhizobium sp. UYSB13 is described as a diazotrophic ACC deaminase-producing isolate, and thus these mechanisms could be involved in the PGP effect observed by this strain.

On the other hand, Pantoea spp. are also reported as PGPB of various crops such as maize, rice, sugarcane, canola, lentil (Lens culinaris) and pea (Pisum sativum) (Verma et al. 2001; Sergeeva et al. 2007; Montañez et al. 2012; Quecine et al. 2012). In the case of the last three crops aforementioned, IAA production has been described as the PGP mechanism (Sergeeva et al. 2007). Moreover, isolate UYSB45 was closely related to the reference species Pantoea dispersa (Fig.1), and its 16S rRNA sequence had 98 % identity with Pantoea dispersa LMG2603 (Table S1). Strain P. dispersa 1A, isolated from a sub-alpine soil in the northwestern Indian Himalayas, showed PGP effects when it was inoculated onto wheat under greenhouse conditions (Selvakumar et al. 2007). This strain was reported to possess several PGP features including IAA production. Our data show that isolate UYSB45 is a diazotroph as well as an IAA producer, so it can be speculated that some of these mechanisms could also be involved in the PGP effect observed by this strain in cv. M81E. Nevertheless, additional studies are needed in both PGPB strains to determine which mechanism is involved in their PGP abilities.

To our knowledge, this is the first work in which an isolate related to the genus Pantoea is reported as a PGPB in sweet sorghum.

Concluding remarks

In this study, a wide variety of putatively endophytic diazotrophs were isolated from the most common commercial sweet sorghum genotype used in Uruguay cv. M81E. All the isolates were biochemically and genetically characterized, with some showing several PGP and infection traits probably involved in plant infection and plant growth promotion. Moreover, two isolates showed PGP effect when they were used as inoculants on sweet sorghum under greenhouse conditions. In addition to known phylotypes, the novel isolates were for the first time described as putative endophytes associated with sweet sorghum as well as PGPBs of this crop. The latter isolates are of potential biotechnological importance, and will be subjected to further PGP characterization in complex systems with the aim of producing an inoculant for sweet sorghum cultivars.