Isolation, Characterization and Plant Growth Promotion Effects of Putative Bacterial Endophytes Associated with Sweet Sorghum (Sorghum Bicolor (L) Moench)

Ann Microbiol (2015) 65:1057–1067 DOI 10.1007/s13213-014-0951-7

ORIGINAL ARTICLE

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

Cintia Mareque & Cecilia Taulé & Martín Beracochea & Federico Battistoni

Received: 21 April 2014 /Accepted: 28 July 2014 /Published online: 13 August 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Sweet sorghum (Sorghum bicolor)iscultivatedin Chryseobacterium, Kocuria, Brevibacillus, Uruguay in complementation with sugarcane (Saccharum Paenibacillus, Bacillus and Staphylococcus.PGPand officinarum) as a feedstock for bioethanol production. It re- infection features were investigated in vitro, and re- quires the application of high levels of chemical fertilizer for vealed some promising biotechnological candidates. In optimal growth, which causes environmental degradation. addition, isolates UYSB13 and UYSB45 showed PGP Plant growth-promoting (PGP) bacteria are of biotechnologi- effects in greenhouse assays. This work provides the cal interest since they can improve the growth of several basis for further studies under field conditions, with important agronomical crops. Of particular interest are endo- the final aim of developing an effective inoculant for phytes, which are those bacteria that can be detected at a sorghum. particular moment within the internal tissues of healthy plants from where they can promote their growth. The Keywords Endophytes . Sweet sorghum . Plant growth aims of this work were to isolate and characterize, as promotion well as identify putatively endophytic bacteria associated with sweet sorghum (cv-M81E), and also to study the inoculation effects of selected isolates on sorghum Introduction growth. A collection of 188 putative endophytes from surface-sterilized stems and roots was constructed and Fossil energy resources are depleting dramatically in characterized. Bacterial isolates were shown to belong order to meet the increasing world energy demands. to different genera including Pantoea, Enterobacter, Moreover, climate change caused by carbon emissions Pseudomonas, Acinetobacter, Stenotrophomonas, from fossil fuels reinforces the need to search for alter- Ralstonia, Herbaspirillum, Achromobacter, Rhizobium, natively energy sources. Crop plants are one of the best sources of renewable energy, as they can be used as Electronic supplementary material The online version of this article feedstock for biofuel production. With this aim, several (doi:10.1007/s13213-014-0951-7) contains supplementary material, complementary crops are cultivated in Uruguay, such as which is available to authorized users. : : : sugarcane (Saccharum officinarum) and sweet sorghum C. Mareque C. Taulé M. Beracochea F. Battistoni (*) (Sorghum bicolor (L.) Moench) (Kim and Day 2011; Microbial Biochemistry and Genomics Department. Instituto de Ratnavathi et al. 2011). Globally, sweet sorghum is the Investigaciones Biológicas Clemente Estable (IIBCE), Avenida Italia 3,318, Montevideo 11600, Uruguay fourth most important cereal, and is known as a multi- e-mail: [email protected] purpose crop since it is used for grain, forage, syrup, C. Mareque fodder and bioethanol production (Almodares and Hadi e-mail: [email protected] 2009; Shoemaker and Bransby 2010). Nevertheless, this C. Taulé crop has a high demand for chemical fertilizer for e-mail: [email protected] optimal productivity, resulting in leaching and run-off M. Beracochea of nutrients, especially nitrogen (N) and phosphorus (P), e-mail: [email protected] leading to environmental degradation (Adesemoye and 1058 Ann Microbiol (2015) 65:1057–1067

Kloepper 2009). These problems emphasize the need for Materials and methods new technologies in agriculture with the aim of attaining more sustainable production systems. A prom- Isolation of putatively endophytic bacteria associated ising alternative to chemical fertilization is the use of with sweet sorghum plant growth-promoting bacteria (PGPB) (Lugtenberg and Kamilova 2009). Amongst these, bacterial endo- With the aim of isolating putative endophytes associated with phytes are referred to as those bacteria that can be the commercial sweet sorghum cv. M81E, two approaches detected at a particular moment within the internal tis- were employed. In the first one, bacteria were isolated from sues of an apparently healthy host plant (Hallmann the roots and stems of trap plants. For this, sterilized seeds et al. 1997;Schulzetal.2006). In contrast to phyto- were sowed into pots containing sterile sand and soil from the pathogenic bacteria, they do not cause any disease sweet sorghum cropping region, Bella Union, Artigas, Uru- symptoms; indeed, they can promote plant growth guay (30°37′56′S, 57°21′18′W, 120 m asl). In the second (James 2000;Berg2009). Mechanisms involved in en- approach, bacteria were isolated from seeds, roots and stems dophytic plant growth promotion (PGP) could be direct of plants collected directly from the field in the same cropping or indirect. Direct PGP mechanisms include biological region. The same surface-sterilization protocol was used for nitrogen fixation (BNF) and mineral solubilisation (P, seed and plant material in both cases. Briefly, 10 g of material Fe), as well as the production of plant phytohormones were incubated for 5 min in 70 % EtOH, then 20 min in 4 % (auxins, cytokinins and gibberellins), while indirect sodium hypochlorite, and finally rinsed 4 times with sterile mechanisms include biocontrol against phytopathogens deionized water. Sterilized seeds for trap plants were germi- mediated by antibiotics, competition for nutrients and nated on 0.8 % water agar plates before sowing. On the other niches, or the induction of an induced systemic resis- hand, for bacterial isolation from roots, stem and seeds, they tance (ISR) response (Rosenblueth and Martínez- were aseptically macerated in a solution of 0.9 % NaCl sup- Romero 2006; Mei and Flinn 2010; Compant et al. plemented with cyclohexamide (100 μg μl−1). Serial dilutions 2010). A number of reports have demonstrated the from the suspensions obtained, were inoculated onto agar association of sweet sorghum with several bacterial en- plates containing DYGs and LGI media in the case of trap dophytes belonging to the genera Herbaspirillum, plants, or TSA (Tryptic Soy Agar; Difco) medium in the case Azospirillum, Klebsiella, Enterobacter, Burkholderia, of field plants. Those culture media were selected with the aim Paenibacillus and others (Olivares et al. 1996; Budi to have a high diversity of heterotrophic bacteria isolates. All et al. 1999; Zinniel et al. 2002; Grönemeyer et al. isolates obtained were purified and stored at −80 °C in 50 % 2011). Moreover, a PGP effect was demonstrated for glycerol. several diazotrophic endophytes, such as Azospirillum lipoferum, A. amazonense, Herbaspirillum seropedicae Screening for biofertilization activities and Gluconacetobacter diazotrophicus, when they were inoculated onto sweet sorghum under greenhouse and With the aim of detecting putatively diazotrophic isolates, the field conditions (Pereira et al. 1988; Sarig et al. 1990; whole collection was subjected to nifH PCR amplification by Chiarini et al. 1998). In consideration of this, the man- using the primers PolF (5′-TGCGAYCCSAARGCBGACTC- agement of the interaction between endophytes and their 3′) and PolR (5′-ATSGCCATCATYTCRCCGGA-3′) (Poly hosts (such as sweet sorghum) might play a significant et al. 2001). In all cases, cell lysates were used as templates role in the development of more sustainable agricultural (Rivas et al. 2001) and a single colony resuspended in water as production systems. In Uruguay, the major sweet sor- a starting material. The PCR mixture was 2.5 μl 10× ghum cultivar used by the producers is cv. M81E, Fermentas Taq reaction buffer, 3.0 mM MgCl2, 0.16 mM which is of national interest. However, until now, no dNTP’s, 0.8 μM of both set of primers, 0.5 U Fermentas studies have been conducted in Uruguay on the native Taq polymerase, 4 % BSA and 4.0 μl of a cell lysate template, microorganisms naturally associated with sweet sorghum or in a final reaction volume of 25 μl. The PCR conditions were have evaluated their potential PGP capability. The aims of this as follows: 1 cycle at 95 °C for 5 min; 30 cycles at 95 °C for work were: (1) to obtain a collection of culturable putatively 45 s; 58 °C for 45 s, and 72 °C for 30 s; and a final cycle at endophytic bacteria associated with sweet sorghum cv. M81E, 72 °C for 5 min. The amplification products were analyzed by (2) to characterize the collection based on PGP and infection 1 % (w/v) agarose gel electrophoresis in TAE buffer and features and thus to identify isolates of interest, and (3) to stained with GoodView (Beijing SBS; Genetech). study the inoculation effects of selected isolates on sweet In addition, the ability to fix N2 was tested in those isolates, sorghum growth. The data obtained will contribute to future which harbored the nifH gene, in vials containing LGI, LGI-P research aimed at developing sweet sorghum inoculants based and JNFb N-free semisolid media (Reis et al. 1994; Perin et al. on native PGPB specifically for cv. M81E. 2006). The vials were incubated at 30 °C for up to 7 days and Ann Microbiol (2015) 65:1057–1067 1059 those which showed a growth pellicle were replicated into a containing 200 μl of TSB (Tryptic Soy Broth; Difco) medium, new fresh vial containing the same media with the aim until an optical density at 620 nm of 0.2 (DO620nm=0.2). The of confirming the presence of the growth pellicle plates were then incubated for 48 h at 30 °C without agitation, (Baldani et al. 2014). the supernatant was removed, and the plates were washed with Inorganic phosphate solubilization ability and siderophore 1× phosphate buffered saline (PBS). For staining, a 0.1 % CV production were tested on plates containing either GL rich solution was added (200 μl per well) and the plates were (Sylvester-Bradley et al. 1982) or chromeazurol medium incubated for 20 min. The excess CV was removed by wash- (Schwyn and Neilands 1987), respectively. After 72 h of ing the plates under running tap water while the bound CV growth, the presence of a translucent or yellow halo around was released from the cells by adding 200 μlof95%EtOH. the colony was evaluated, indicating phosphate solubilizing or The absorbance of the suspension was measured at siderophore-producing isolates, respectively. In the case of the 570 nm. All steps were carried out at room temperature siderophore production assay, Herbaspirillum seropedicae (Peeters et al. 2008). Z67 was used as a positive control (Rosconi et al. 2013). Evaluation of bacterial genomic diversity by ERIC-PCR Production of enzymes for phytostimulation With the aim of evaluating the genomic diversity of the An assay for the quantitative detection of indole-3-acetic acid selected isolates associated with sweet sorghum cv. M81E, (IAA) production was performed on all the bacterial collection the Enterobacterial Repetitive Intergenic Consensus PCR by using a colorimetric method (Sarwar and Kremer 1995)as (ERIC-PCR) technique was employed as described previous- described previously (Taulé et al. 2012). ly (Taulé et al. 2012). Data were further analyzed using With the aim of detecting aminocyclopropane-1- GelComparII6.5 (Applied Maths) software. The similarity carboxylate (ACC) deaminase activity, all the isolates were between strains was expressed by Dice’s coefficient, and grown overnight at 28 °C in a 96-microwell plate containing cluster analysis was performed using the unweighted pair 2 ml of LGI+N medium to mid- to late-log phase. Cultures group average linkage method (UPGMA) with a similarity were harvested by centrifugation at 4,000 g for 20 min, the of 80 %. supernatants were removed and the pellets were washed twice with 1 ml of LGI−N medium. The washed cells were resus- 16S rRNA gene amplification, sequencing and phylogenetic pended in 150 μl of LGI N-free medium and an aliquot of 5 μl analysis was inoculated onto plates containing solid LGI N-free plates complemented with 5 % N and 30 mmol plate−1 of ACC Amplification and sequencing of the 16S rRNA gene was (Penrose and Glick 2003). All plates were supplemented with performed for selected isolates as described before (Taulé 1.8 % Bacto-Agar (Difco). Plates were incubated for 3 days at et al. 2012). The 16S rRNA gene sequences were deposited 30°C.Isolates,whichwereabletogrowthwithACCasasole in GenBank with the following accession numbers: source of N, were considered as positive. KJ532081–KJ532123. The quality of the sequences obtained was checked manu- Screening for hydrolytic activity ally and assembled using DNA Baser Sequence Assembler v.3.× (2010) (http://www.dnabaser.com). Sequences were For determination of hemicellulose, cellulose and peroxidases imported into the ARB software package v.5.5 (Ludwig activities, the bacterial collection was grown in plates contain- et al. 2004) and added to the database. Sequences were aligned ing TSA solid medium supplemented with 0.5 % Avicel using ARB FastAligner, then refined manually. Phylogenetic (Sigma-Aldrich, Germany) 0.2 % cellulose or 250 mg l−1 trees were generated using the maximum parsimony, neighbor ABTS (Sigma-Aldrich) with and without the addition of joining and maximum likelihood algorithms with 1,000 boot- −1 0.1gl MnCl2·4H2O, respectively. In the case of protease strap replicates. activity detection, plates containing TSA medium were supplemented with 5 % (w/v) of skimmed milk (Hofrichter and Capacity of the isolates to grow using different C, N sources Fritsche 1997; Kim et al. 2008; Martinez-Rosales and Castro- and with various antibiotics Sowinsky 2011). The entire bacterial collection was grown on plates containing Screening for biofilm formation LGI medium supplemented with the C or N sources to be tested at the final concentrations as the original protocol Biofilm formation was detected using the crystal violet (CV) (Cavalcante and Dobereiner 1998). The C-sources analyzed method (Christensen et al. 1985). For this, the collection was were maltose, mannitol, glucose, sucrose, malate, fructose, first grown at 30 °C with agitation, in 96-microwell plates lactose, sucrose, glycerine, pyruvic acid and vinasse 1060 Ann Microbiol (2015) 65:1057–1067

(byproduct generated during the distillation of ethanol, yeast, sterilized material, they can be considered to be putative amino acids and/or organic acids from cane molasses fermen- endophytes until proven by microscopy. The plant material tation mixtures); the last two were obtained from industrial was collected from a crop region in which plants were grown waste. In the case of the N-sources tested, these were with low level of N fertilization. With the aim of isolating a

(NH4)2SO4,KNO3,NH4Cl, L-tyrosine, L-asparagine and L- broad range of bacteria, various isolation approaches (from glutamic acid as well as urea. trap plants and field material), as well as different bacterial For antibiotic resistance determination, TSA plates were growth media (DYGs, LGI and TSA) were employed. With supplemented with gentamicin, kanamycin, streptomycin, regard to the growth media, 62, 44 and 82 isolates were spectinomycin, ampicillin, polymixina b, tetracycline or isolated from DYGs, LGI and TSA media, respectively. The nalidixic acid, all at a final concentration of 100 μlml−1,as genomic diversity of the entire bacterial collection was ana- well as neomycin at a final concentration of 25 μlml−1. lyzed by ERIC-PCR, and 6 different groups with a similarity value of 80 % were found using the GelComparII6.5 software Plant growth promotion of sweet sorghum inoculated (Table 1). In addition, 58 putatively diazotrophic isolates were with selected isolates detected in the collection by PCR amplification of the nifH gene (Table 1). These latter isolates were tested for their The growth response of sweet sorghum to bacterial inocula- availability to grow in LGI, LGI-P and JNFb semi-solid N- tion was studied in greenhouse conditions. Seeds were surface free media. Using this approach, 38 isolates capable of pro- sterilized as described above and incubated for 45 min with ducing a growth pellicle were distinguished (Table 1). slow agitation with a suspension of 1.0 x108 cells ml−1 of each isolate to be tested. Inoculated seeds were germinated on Characterization of sweet sorghum associated isolates 0.8 % water agar for 2 days, transferred to pots containing 1.5kgofsand:soil(1:2)assubstrateandmaintainedingreen- With the aim of detecting plant growth promotion features, the house conditions with a photoperiod of 8/16 h light/dark. whole collection was screened for the ability to produce IAA, After 30 days post-inoculation (pi), plants were re-inoculated. siderophores, solubilise phosphates and the presence of ACC- This experiment had 5 treatments with 10 replicates in a deaminase activity. Out of 188 isolates tested, 33 and 18 were completely randomized design. The isolates tested as inocu- able to produce IAA and siderophores, respectively, 22 to lants were Rhizobium sp. UYSB12, Bacillus sp. UYSB13, solubilize phosphate, while 130 presented ACC activity (Ta- Enterobacter sp. UYSB34 and Pantoea sp. UYSB45. As a ble 1). In addition, the collection was also screened for the negative control, a treatment containing plants without inocu- presence of plant infection traits, including hemicelluloses, lation was employed. Additionally, plants inoculated with the cellulose, protease, peroxidase activities and biofilm forma- known PGPB strain Azospirillum brasilense Sp7 were used as tion. Results showed that only 3 and 5 isolates presented a positive control reference treatment. At 3 months pi, the hemicellulase and cellulase activity, respectively, while 26 experiment was harvested, roots and aerial parts were dried at and 41 isolates presented protease activity and biofilm forma- 60 °C until constant weight, and their dry weights then tion ability, respectively (Table 1). determined. Physiological features of the bacterial isolates Statistical analysis The ability to grow in different C and N sources, as well as in Statistic analyses were done with the Infostat programme, and the presence of antibiotics in the culture media, was evaluated in those treatments where significant differences were for the entire bacterial collection. As sole C-source, 97 % of confirmed the Fisher LSD test (p<0.10) was employed the isolates were able to grow in fructose or crystal sugar, (InfoStat 2008). 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, Results 93 % in L-tyrosine, 89 % in urea, 88 % in L-glutamic acid,

63 % in (NH4)2SO4 and none were able to grow in the Isolation of putatively endophytic diazotrophic bacteria presence of NH4Cl (Table S2). associated with sweet sorghum cv. M81E When the capacity to grow in the presence of various antibiotics was evaluated, it was found that 100% of the A library containing 188 isolates from surface sterilized sweet isolates were able to grow in the presence of neomycin, sorghum seeds (2 isolates), stems (79 isolates) and roots (107 98 % in ampicillin, 96 % in spectinomycin, 89 % in strepto- isolates) was constructed. As they were isolated from surface- mycin, 81 % tetracycline, 77 % in gentamycin, 66 % in Ann Microbiol (2015) 65:1057–1067 1061

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

Isolate ERIC group Identificationa Phytostimulationb Biofertilizationc Infectiond

IAA ACC nifH GP Ca3PO4 SID CE HC PROT Biofilm formation

UYSB01 2 Ralstonia −− ++− + −−− − UYSB02 2 Ralstonia − ++−− + −−− − UYSB03 4 Staphylococcus −− + −− + −−− − UYSB04 3 Bacillus −− + −− −−−++ UYSB05 2 Enterobacter −− ++−−−−−− UYSB06 2 Bacillus − +++−−−−−− UYSB07 2 Bacillus − ++−− −+ −− − UYSB08 1 Kocuria − +++−−−−−− UYSB09 1 Achromobacter − + −−− −−−− − UYSB11 2 Rhizobium − +++−−−−−+ UYSB12 2 Rhizobium − +++−−−−++ UYSB13 2 Rhizobium − +++−−−−++ UYSB14 2 Ralstonia −− + − ++−−− − UYSB15 2 Bacillus − +++−−−−−− UYSB17 2 Rhizobium − ++−− −−−− + UYSB18 3 Bacillus −− −−+ −−−+ − UYSB19 2 Paenibacillus − +++−−−−−+ UYSB20 2 Bacillus − ++ND−−−−−− UYSB21 2 Pantoea ++ ++− + −−− − UYSB22 2 Enterobacter ++ ++− + −−− − UYSB23 2 Rhizobium − ++−− + −−++ UYSB25 2 Rhizobium − ++−− −−−− − UYSB26 5 Bacillus + −−−+ −−−− + UYSB27 2 Paenibacillus −− + −− −−−− + UYSB28 2 Brevibacillus − +++−−++−− UYSB29 2 Brevibacillus ++ +− + −−−− − UYSB30 2 Acidovorax ++ −−− + −−− − UYSB32 2 Stenotrophomonas −− +++ +−−++ UYSB33 2 Stenotrophomonas ++ ++−−−−−+ UYSB34 2 Enterobacter ++ ++−−−−−− UYSB35 2 Herbaspirillum − + −−++−−− + UYSB36 5 Brevibacillus ++ +−− −−−− − UYSB37 5 Pseudomonas ++ −−+ −−−− + UYSB38 2 Pantoea ++ −−+ −−−− − UYSB39 2 Pantoea + − − −− −−−− + UYSB40 2 Stenotrophomonas + − + −− −−−++ UYSB41 5 Acinetobacter − + −−− −++−− UYSB42 2 Acinetobacter + − + − + −−−− − UYSB43 3 Pantoea ++ −−+ −−−− + UYSB45 3 Pantoea + − +++ +−−− − UYSB46 1 Chryseobacterium ++ −−++−−− + UYSB47 2 Pantoea − + −−+ − + −− + UYSB48 3 Serratia ND ND + ND ND − ND ND + ND a Taxonomic identification based on 16S rDNA gene similarity b IAA indole-3-acetic acid production; ACC aminocyclopropane-1-arboxylate deaminase activity c nifH gene detected by PCR approach; GP growth pellicle; Ca3PO4 solubilization of inorganic calcium phosphate; SID siderophore production d CE exo-cellulase activity; HC hemicellulase activity; PROT protease activity 1062 Ann Microbiol (2015) 65:1057–1067 polymyxin B, 63 % in nalidixic acid and 43 % in kanamycin isolates, and grouped together in a cluster closely relat- (Table S2). ed to the reference species Stenotrophomonas maltophilia. Identification and phylogenetic analysis of sweet sorghum Betaproteobacteria spp. were also present in the collection associated isolates based on their partial 16S rRNA sequences and they grouped in a well supported cluster with three different branches (Fig. 1). Isolate UYSB09, which belonged For 16S rRNA gene sequencing and analysis, isolates were to the genus Achromobacter (Fig. 1; Table S1), did not selected according to their in vitro PGP features and their ERIC genomic patterns. As shown in Table S1, BLAST

T 56 Pantoea dispersa ,DQ504305 searches against the NCBI database revealed close relation- 70 UYSB45 61 UYSB43 ships to known plant-associated bacteria, including genera 53 Pantoea wallisiiT, JF295057 66 UYSB39 T 61 Pantoea ananatis , U80196 Pantoea stewartii subsp. stewartiiT, U80208 belonging to the phyla Alphaproteobacteria (Rhizobium), 92 UYSB38 100 88 UYSB05 Betaproteobacteria (Achromobacter, Herbaspirillum and 96 UYSB34 99 UYSB22 Enterobacter oryzaeT, EF488759 99 T Ralstonia), Gammaproteobacteria (Acinetobacter, Enterobac- 66 Enterobacter radicincitans , AY563134 Serratia marcescens subsp. marcescensT, 91 100 UYSB48 T ter, Pantoea, Pseudomonas, Serratia and Stenotrophomonas), 100 Acinetobacter calcoaceticus , AJ888983 99 Acinetobacter pittiiT, HQ180184 Actinobacteria (Kocuria), Firmicutes (Brevibacillus, Bacillus, 100 UYSB42 100 Acinetobacter brisouiiT, DQ832256 Acinetobacter parvusT, AJ293691 Paenibacillus,andStaphylococcus) and Bacterioidetes 99 UYSB41 T 70 84 Pseudomonas fulva , AB046996 (Chryseobacterium). 99 Pseudomonas putidaT, D84020 100 UYSB37 Pseudomonas psychrotoleransT, AJ575816 99 UYSB01 A phylogenetical tree (dendrogram) was constructed based 98 99 UYSB02 84 Ralstonia pickettiiT, AY741342 upon the 16S rRNA gene sequences of 34 selected isolates 100 Ralstonia insidiosaT, AF488779 100 Ralstonia solanacearumT, EF016361 Cupriavidus necatorT, AF191737 100 T (Fig. 1). Since the largest number of isolates associated with 82 Cupriavidus taiwanensis , AF300324 T 93 Herbaspirillum rubrisubalbicans , AF137508 99 Herbaspirillum seropedicaeT, Y10146 sweet sorghum belonged to the Gammaproteobacteria, this 100 T 100 Herbaspirillum frisingense , AJ238358 UYSB35 T 97 Achromobacter marplatensis , EU150134 group will be described first. As can be seen from Fig. 1, Achromobacter xylosoxidans subsp. xylosoxidansT, 100 UYSB09 Pantoea isolates UYSB38, UYSB39, UYSB43, and UYSB45 50 59 95 UYSB32 84 UYSB33 100 Stenotrophomonas maltophiliaT, AB294553 grouped in a cluster with two branches, within which isolates 97 Stenotrophomonas pavaniiT, FJ748683 Stenotrophomonas daejeonensisT, 100 Xanthomonas albilineansT, X95918 UYSB39 and UYSB45 clustered closely with the reference 99 Xanthomonas sacchariT, Y10766 T 79 Rhizobium radiobacter , AB247615 80 UYSB13 strains P. wa ll is ii and P. dispersa,respectively,butnoneofthe T 95 81 Rhizobium skierniewicense , HQ823551 98 UYSB12 other isolates clustered close to any reference strains. The 16S 100 Rhizobium pusenseT, FJ969841 85 UYSB11 Rhizobium rosettiformansT, EU781656 rRNA sequences of isolates UYSB05, UYSB22 and UYSB34 100 55 UYSB25 99 Rhizobium mesosinicumT, DQ100063 T 100 Rhizobium alamii , AM931436 showed that they belonged to the genus Enterobacter (Fig. 1, Rhizobium sullaeT, Y10170 T 72 Chryseobacterium formosense , AY315443 UYSB46 100 Table S1) and that they grouped in one well supported cluster Chryseobacterium defluviiT, AJ309324 95 Bacillus megateriumT, D16273 distant from any reference strain. In addition, isolate 85 UYSB26 100 Bacillus flexusT, AB021185 UYSB18 58 T UYSB48, which has an identity of 99 % with Serratia 62 80 Bacillus niacini , AB021194 56 UYSB20 T 66 99 Bacillus pocheonensis , AB245377 marcescens WW4 (Table S1), shared the same node as UYSB06 T 100 Bacillus acidiceler , DQ374637 UYSB07 Enterobacter spp., but was located in a different branch clus- 100 T 100 Bacillus luciferensis , AJ419629 T 64 Bacillus pumilus , AY876289 tered closely with the reference species Serratia marcescens 78 UYSB15 100 Bacillus safensisT, AF234854 Bacillus altitudinisT, AJ831842 subsp. marcescens. 59 58 99 UYSB03 Staphylococcus epidermidisT, D83363 100 T 100 Staphylococcus aureus subsp. aureus , Pseudomonas and Acinetobacter spp. shared the same 71 Brevibacillus laterosporusT, D16271 100 UYSB36 UYSB29 node, and clustered in two separated branches. With 100 T 100 Brevibacillus brevis , AB271756 100 Brevibacillus panacihumiT, EU383033 T regard to the Acinetobacter spp., isolate UYSB41 clus- 55 Paenibacillus pabuli , AB045094 100 Paenibacillus taichungensisT, EU179327 UYSB19 tered closely related with the reference strain A. parvus, 100 100 UYSB27 Paenibacillus glycanilyticusT, AB042938 100 Paenibacillus prosopidisT, FJ820995 T while isolate UYSB42 grouped in a different branch 98 Kocuria palustris , Y16263 74 UYSB08 T 100 Kocuria varians , X87754 distant from any reference strain. The only isolate relat- Kocuria flavaT, EF602041 ed to the genus Pseudomonas in the tree, UYSB37 Thermaerobacter litoralisT, AY936496 0.02 (Table S1;Fig.1), clustered in a branch distant from Fig. 1 Neighbor-joining phylogenetic tree based on bacterial 16S rRNA any reference strain. sequences of representative isolates. The tree shows the phylogenetic Finally, from the Gammaproteobacteria phylum, iso- affiliation of 34 partial 16S rRNA sequences of endophytic bacterial lates UYSB32 and UYSB33, which belong to the genus isolated from sweet sorghum (Sorghum bicolor). Numbers at branches represent bootstrap values >50 % from 1,000 replicates. Thermaerobacter Stenotrophomonas (Table S1), were located in a branch litoralis was used as an outgroup. The scale bar shows the number of totally separate from the other Gammaproteobacteria nucleotide substitutions per site Ann Microbiol (2015) 65:1057–1067 1063 group with any reference strain, but shared the same node as reference strain, isolate UYSB36 was closely related to the the reference strains A. xylosoxidans,andA. marplatensis. reference species B. laterosporus. Additionally, isolates UYSB01 and UYSB02, which on the Finally, from the Firmicutes, isolates UYSB19 and basis of their 16S rRNA gene sequences were similar to UYSB27, which belonged to the genus Paenibacillus Ralstonia spp. (Table S1), grouped together in a cluster (Table S1), were grouped in a well-supported cluster (Fig.1); distant from any reference strain (Fig. 1). Finally, from the isolate UYSB27 grouped closely with the reference species Betaproteobacteria, the only isolate represented in the genus P. glycanilyticus while isolate UYSB19 grouped in a separate Herbaspirillum UYSB35, although it clustered together with branch distant from any reference strain. Herbaspirillum spp., it was in a separate branch distant from Finally, and with regard to the only isolate from the any reference strain (Fig. 1). Actinobacteria (Table S1), the dendrogram based on 16S With regard to the Alphaproteobacteria (Fig. 1), the 16S rRNA sequences shows that isolate UYSB08 grouped closely rRNA sequences of isolates UYSB11, UYSB12, UYSB13, with the reference species Kocuria palustris. 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 Plant growth promotion of sweet sorghum inoculated strains R. pusense, R. skierniewicense and R. mesosinicum, with selected bacterial isolates under greenhouse conditions respectively, while isolate UYSB11 grouped alone distant from any reference strain. Isolates tested as inoculants on sweet sorghum were The only isolate in the Bacterioidetes phylum, UYSB46, selected from the bacterial collection according to their grouped closely with the reference species Chryseobacterium in vitro PGP features and their phylogenetic affiliations. formosense (Fig. 1). Inoculated plantlets were grown under greenhouse con- The Firmicutes was well represented in the collection ditions in pots containing sterile sand and soil as a (Table S1), and the dendogram based on 16S rRNA sequences substrate. Four-month pi plants were harvested and bio- showed that isolates from this class grouped in a single cluster metric parameters as well as total N content (%) were (Fig. 1). The genus Bacillus was represented in the collection measured (Table 2). None of the isolates showed any by the isolates UYSB06, UYSB07, UYSB15, UYSB18, significant differences from the control when stem di- UYSB20, and UYSB26 (Table S1). Isolates UYSB07, ameter was evaluated, but isolates Rhizobium sp. UYSB15, UYSB20, and UYSB26 grouped closely related UYSB13 and Pantoea sp. UYSB45 showed significant with the reference species B. acidiceler, B. pumilus, B. niacini differences from the negative control in their stem and B. megaterium, respectively, while the remaining isolates height and dry weight (roots and shoots) (Table 2). clustered distant from any reference strain. In addition, the only Additionally, isolates Rhizobium sp. UYSB12 and isolate representing the genus Staphylococcus UYSB03, shared Enterobacter sp. UYSB34 showed significant differ- thesamenodeastheBacillus isolate UYSB15, and grouped ences from the control only in their root and the shoot closely with the reference strain Staphylococcus epidermidis. dry weights, respectively. Finally, A. brasilense Sp7, With regard to the genus Brevibacillus, isolates UYSB29 which was used as a reference strain, did not show and UYSB36 (Table S1) grouped in a single well-supported any significant differences from the negative control in cluster (Fig.1), while isolate UYSB29 did not cluster with any all the parameters evaluated.

Table 2 Effects of inoculation a −1 with putative bacterial endo- Treatment Stem height (cm) Stem diameter (mm) Dry weight (g plant ) phytes on the growth of sweet sorghum cv. M81E Roots Shoot

Negative Control 13.50 a 4.84 a 0.74 a 0.86 a a Negative controls are uninocu- Rhizobium sp. UYSB12 14.44 ab 4.90 a 1.05 c 0.95 abc lated plants or with N-fertilization added Rhizobium sp. UYSB13 16.00 b 4.44 a 1.09 c 1.07 cd b Means within two treatments Enterobacter sp. UYSB34 14.95 ab 4.73 a 0.93 abc 1.02 bcd that have the same letter are not Pantoea sp. UYSB45 16.25 b 4.79 a 1.01 bc 1.15 d significantly different by LSD Azospirillum brasilenseSp7 14.30 ab 4.90 a 0.77 ab 0.94 ab 0.10 test 1064 Ann Microbiol (2015) 65:1057–1067

Discussion of Namibia (Grönemeyer et al. 2011). To our knowledge, this is the first report in which isolates of the genera Pantoea, A collection containing 188 isolates associated with roots and Acinetobacter and Stenotrophomonas were described as being stems of the commercial sweet sorghum cv. M81E was con- associated with sweet sorghum. structed and characterized. Out of these, 57 isolates were The Alphaproteobacteria was represented in this study by nifH+ from which 52 were able to produce a growth pellicle strains of the genus Rhizobium. The phylogenetic analysis in semi-solid N-free media, and so they were defined as revealed two different groups within this genus: one closely diazotrophs. In addition, a number of isolates with in vitro related to the phytopathogen R. radiobacter and the non- biofertilization, phytostimulation and infection features were nodulated R. pusense reference strains, while the other group also detected in the collection, suggesting their biotechnolog- was more closely related to plant-associated or nodulating ical potential as inoculants for this cultivar. Phylogenetic Rhizobium species including R. mesosinicum, R. alamii and analysis revealed a high diversity of bacteria associated with R. sullae. There have been several reports of the isolation of sweet sorghum including the genus Rhizobium from the rhizobia as endophytes and/or associated with various non- Alphaproteobacteria, the genera Achromobacter, legume crops (Hallmann et al. 2006; Rosenblueth and Martí- Herbaspirillum and Ralstonia from the Betaproteobacteria, nez-Romero 2006; Taulé et al. 2012; Beneduzi et al. 2013), and a high number of isolates related to the but until the present study none came from sweet sorghum. Gammaproteobacteria, including the genera Pseudomonas, The genera Ralstonia, Achromobacter,andHerbaspirillum of Acinetobacter, Pantoea, Enterobacter and Stenotrophomonas. the Betaproteobacteria were also present in our collection. In addition, an isolate belong to the Bacterioidetes was detect- Some species of the genus Ralstonia have been reported to ed, sharing 98 % 16S rRNA sequence identity with the refer- be associated with roots of sweet sorghum from Namibia as ence strain Chryseobacterium formosense CC-H3-2. Interest- well as being endophytes of soybean (Kuklinsky-Sobral et al. ingly, Gram-positive isolates were also detected, including 2005; Grönemeyer et al. 2011). Species of the genus one isolate belong to the Actinobacteria, which was related Achromobacter were previously described as putative endo- to the genus Kocuria, and 13 isolates belonging to the phytes associated with Citrus spp., sunflower (Helianthus Firmicutes, which were related to the genera Brevibacillus, annuus) and sugarcane, as well as rhizobacteria associated Paenibacillus, Staphylococcus and Bacillus. with canola (Brassica napus) (Araújo et al. 2001;Bertrand In this study, the plants used for bacterial isolation were et al. 2001; Ambrosini et al. 2012; Taulé et al. 2012). In healthy, but some of the genera identified have been reported addition Herbaspirillum spp. have been isolated and reported as phytopathogens, such as the plant-associated R. radiobacter as associated (including endophytically) with various crops, (formerly Agrobacterium tumefaciens), which is the causal such as rice (Oryza sativa), maize (Zea mays), sugarcane and agent of crown gall. Moreover, several isolates were of clinical sweet sorghum (Olivares et al. 1996;Monteiroetal.2012), importance, such as Stenotrophomonas maltophila, Pseudo- but to our knowledge there are no previous reports of monas putida, Staphylococcus epidermidis and Ralstonia spp. Achromobacter and Rhizobium species associated with sweet Nevertheless, most of the genera identified have been reported sorghum plants. as being associated with a number of important agronomical The Bacterioidetes phylum was represented by only one crops including sweet sorghum (Hallmann et al. 2006; isolate, which belongs to the genus Chryseobacterium.Spe- Rosenblueth and Martínez-Romero 2006). The most abundant cies of this genus were described as endophytes of several and diverse phylum of bacteria in the collection was the plants, such as cucumber (Cucumis sativus), potato (Solanum Gammaproteobacteria. Interestingly, the same results were tuberosum), canola and tomato (Solanum lycopersicum) obtained from a collection of putative endophytes associated (Hallmann and Berg 2006), but there are no previous reports with sugarcane varieties cultivated in Uruguay (Taulé et al. of them being associated with sweet sorghum. 2012). Within the Gammaproteobacteria, isolates of the gen- With regarding to the Gram-positives, there were isolates era Enterobacter, Pantoea (Enterobacteriales), Pseudomonas, related to the Actinobacteria (Kocuria spp.) and Firmicutes Acinetobacter (Pseudomonales), and Stenotrophomonas (Paenibacillus, Brevibacillus, Staphylococcus and Bacillus (Xanthomonadales) have been previously reported as being spp.). Strains from the genus Bacillus are well reported as endophytes and/or associated with different Poaceaous crops endophytes and/or as associated bacteria to several crops (Hallmann et al. 2006; Rosenblueth and Martínez-Romero (Hallmann and Berg 2006; Rosenblueth and Martínez- 2006;Beneduzietal.2013). In particular, Enterobacter and Romero 2006). In particular, Bacillus spp. were reported as Pseudomonas isolates were reported as associated with sweet isolated from the rhizosphere of sorghum plants and described sorghum plants from the state of Nebraska in the USA as biocontrol agents, but not as endophytes or PGPB of this (Zinniel et al. 2002), but no isolates belonging to the crop (Budi et al. 1999;Martinez-Absalonetal.2012). In the Gammaproteobacteria were described in a collection of bac- case of Paenibacillus spp., isolates were described as endo- teria associated with sweet sorghum from the Kavango region phytes of cucumber, sweet potato (Ipomoea batatas), and Ann Microbiol (2015) 65:1057–1067 1065 sweet sorghum (Hallmann and Berg 2006; Rosenblueth and 2012). In the case of the last three crops aforementioned, IAA Martínez-Romero 2006;Grönemeyeretal.2011). Regarding production has been described as the PGP mechanism the genus Staphylococcus, numerous isolates were reported as (Sergeeva et al. 2007). Moreover, isolate UYSB45 was close- endophytes associated with various plants, such as carrots ly related to the reference species Pantoea dispersa (Fig.1), (Daucus carota), sugarcane and Alyssum bertolonii (Surette and its 16S rRNA sequence had 98 % identity with Pantoea et al. 2003; Barzanti et al. 2007; Velázquez et al. 2008). dispersa LMG2603 (Table S1). Strain P.dispersa 1A, isolated Additionally, Brevibacillus spp. have also been reported as from a sub-alpine soil in the northwestern Indian Himalayas, endophytes associated with maize and cotton wood (Populus showed PGP effects when it was inoculated onto wheat under deltoides), as well as in balloon flower plants (Platycodon greenhouse conditions (Selvakumar et al. 2007). This strain grandiflorum) (Asraful Islam et al. 2010;Grönemeyeretal. was reported to possess several PGP features including IAA 2011; Brown et al. 2012), but there are no previous reports of production. Our data show that isolate UYSB45 is a strains from these genera associated with sweet sorghum. diazotroph as well as an IAA producer, so it can be speculated Finally, from the Actinobacteria, the literature shows that that some of these mechanisms could also be involved in the Kocuria spp. were isolated from marigolds (Tagetes spp.) and PGP effect observed by this strain in cv. M81E. Nevertheless, reported as biocontrol agents (Sturz and Kimpinski 2004), but additional studies are needed in both PGPB strains to deter- to our knowledge this is the first report in which an isolate mine which mechanism is involved in their PGP abilities. from this genus was reported as a putative endophyte associ- To our knowledge, this is the first work in which an isolate ated with sweet sorghum. related to the genus Pantoea is reported as a PGPB in sweet Thus, our study reveals several novel isolates associated sorghum. with sweet sorghum with plant growth-promoting character- istics, and which are, therefore, excellent potential candidates Concluding remarks for biotechnological application as a PGP inoculant. In this study, a wide variety of putatively endophytic Plant growth promotion of putative endophytes associated diazotrophs were isolated from the most common commercial with sweet sorghum sweet sorghum genotype used in Uruguay cv. M81E. All the isolates were biochemically and genetically characterized, Isolates tested as an inoculant on cv. M81E of sweet sorghum with some showing several PGP and infection traits probably were selected from the collection taking into consideration involved in plant infection and plant growth promotion. More- their PGP features as well as their 16S rRNA-based identity. over, two isolates showed PGP effect when they were used as The results demonstrated that isolates Rhizobium sp. UYSB13 inoculants on sweet sorghum under greenhouse conditions. In and Pantoea sp. UYSB45 were PGPB under the conditions addition to known phylotypes, the novel isolates were for the tested (Table 2). In the present study, Azospirillum brasilense first time described as putative endophytes associated with Sp7, which is well reported as a PGP of sorghum (Steenhoudt sweet sorghum as well as PGPBs of this crop. The latter and Vanderleyden 2000;Lucyetal.2004; Bashan and Luz isolates are of potential biotechnological importance, and will 2010), was also tested as inoculant in the PGP assays, but no be subjected to further PGP characterization in complex sys- significant PGP effects were obtained for this strain in any of tems with the aim of producing an inoculant for sweet sor- the parameters measured under the studied conditions (Ta- ghum cultivars. ble 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 Acknowledgments This work was supported by grants from the Sec- assays (Long et al. 2008). torial Energy Fund (Project FSE_2011_1_5911), of the Uruguayan Na- Rhizobia,suchasRhizobium leguminosarum, tional Agency for Innovation and Research (Agencia Nacional de Bradyrhizobium japonicum and Sinorhizobium meliloti,are Innovación e Investigación-ANII), and the Uruguayan Program for the Development of the Basic Sciences (Programa de Desarrollo de las often reported as PGPB in non-legume plants, such as maize, Ciencias Básicas-PEDECIBA). The authors are very grateful to Ing. rice and sweet sorghum (Rashad et al. 2001; Matiru and Agr. Fernando Hackembruch from the Agriculture Department of the Dakora 2004; Bhattacharjee et al. 2008). In this study, Alcoholes Uruguay S.A. (ALUR S.A.). Rhizobium sp. 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