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Genetic diversity and plant growth promoting traits of diazotrophic bacteria isolated from two Pennisetum purpureum Schum. genotypes grown in the field

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Abstract

Background and aims

Some elephant grass (Pennisetum purpureum) genotypes are able to produce large amounts of biomass and accumulate N derived from BNF when growing in soil with low N levels. However, information about the diazotrophic bacteria colonizing this C4 plant is still very scarce. This study aimed to characterize the plant growth promoting traits of a fraction of culturable diazotrophs colonizing the genotypes CNPGL F06-3 and Cameroon.

Methods

A total of 204 isolates were obtained from surface sterilized leaves, stems and roots after culturing on five different N-free semisolid media. These were then analyzed by BOX-PCR, and the 16S rRNA and nifH sequences of representative isolates were obtained. The functional ability of the isolates to reduce acetylene, produce indole and to solubilize phosphate was also determined.

Results

The diazotrophic bacterial population varied from 102 up to 106 bacteria g−1 fresh tissues of both genotypes. The BOX-PCR analysis suggested a trend in the genetic diversity among the 204 diazotrophic strains colonizing the different genotypes and plant tissues. Sequencing of 16S rRNA fragments confirmed the presence of Azospirillum brasilense and Gluconacetobacter diazotrophicus and revealed for the first time the occurrence of G. liquefaciens, G. sacchari, Burkholderia silvatlantica, Klebsiella sp., Enterobacter cloacae and E. oryzae in elephant grass. Interestingly, several nifH sequences from isolates identified as G. liquefaciens and G. sacchari showed homologies with nifH sequences of Enterobacter species. The majority of the isolates (97%) produced indole compounds, 22% solubilized phosphate and 6.4% possessed both characteristics.

Conclusions

The results showed the occurrence of novel diazotrophic bacterial species colonizing different tissues of both genotypes of elephant grass. In addition, the study revealed the presence of several bacteria with growth promoting traits, and highlighted their potential to be exploited as biofertilizers.

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References

  • Ahemad F, Ahmad I, Khan MS (2008) Screening of free-living rhizobacteria for their multiple plant growth promoting activities. Microbiol Res 163:173–181

    Article  Google Scholar 

  • Albino U, Saridakis DP, Ferreira MC, Hungria M, Vinuesa P, Andrade G (2006) High diversity of diazotrophic bacteria associated with the carnivorous plant Drosera villosa var. villosa growing in oligotrophic habitats in Brazil. Plant Soil 287:199–207

    Article  CAS  Google Scholar 

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Andrade AC, Fonseca DM, Lopes RS, Nascimento Júnior D, Cecon PR, Queiroz DS, Pereira DH, Reis ST (2005) Análise de crescimento do capim-elefante ‘napier’ adubado e irrigado. Ciênc agrotec 29:415–423

    Article  Google Scholar 

  • Baldani JI, Baldani VLD (2005) History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience. An Acad Bras Cienc 77:549–579

    Article  PubMed  CAS  Google Scholar 

  • Baldani JI, Baldani VLD, Dobereiner J (2005) Genus III. Herbaspirillum. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds) Bergey’s manual of systematic bacteriology, 2nd edn. Springer, Newark, pp 629–636

    Chapter  Google Scholar 

  • Bashan Y, Bashan LE (2010) How the plant growth-promoting bacterium Azospirillum promotes plant growth-a critical assessment. Adv Agron 108:77–136

    Article  CAS  Google Scholar 

  • Boddey RM (1987) Methods for quantification of nitrogen fixation associated with gramineae. Crit Rev Plant Sci 6:209–266

    Article  CAS  Google Scholar 

  • Boddey RM (1995) Biological nitrogen fixation in sugar cane: a key to energetically viable biofuel production. Crit Rev Plant Sci 14:263–279

    Google Scholar 

  • Couillerot O, Poirier MA, Prigent-Combaret C, Mavingui P, Caballero-Mellado J, Moënne-Loccoz Y (2010) Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize. J Appl Microbiol 109:528–538

    PubMed  CAS  Google Scholar 

  • Crozier A, Kamiya Y, Bishop G, Yokota T (2000) Biosynthesis of hormones and elicitor molecules. In: Buchanan B, Gruissem W, Jones R (Ed.) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists pp 850–929.

  • Dastager SG, Deepa CK, Puneet SC, Nautiyal CS, Pandey A (2009) Isolation and characterization of plant growth-promoting strain Pantoea NII-186. From Western Ghat forest soil, India. Lett Appl Microbiol 49:20–25

    Article  PubMed  CAS  Google Scholar 

  • Dobbelaere S, Vanderleyden J, Okon Y (2003) Plant growth promoting effects of diazotrophs in the rhizosphere. Crit Rev Plant Sci 22:107–149

    Article  CAS  Google Scholar 

  • Döbereiner J (1988) Isolation and identification of root associated diazotrophs. Plant Soil 110:207–212

    Article  Google Scholar 

  • Döbereiner J (1995) Isolation and identification of aerobic nitrogen-fixing bacteria from soil and plants. In: Alef K, Nannipieri P (eds) Methods in applied soil microbiology and biochemistry. Academic, London, pp 134–141

    Google Scholar 

  • Eckert B, Weber OB, Kirchhof G, Halbritter A, Stoffels M, Hartmann A (2001) Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus. Int J Syst Evol Micr 51:17–26

    CAS  Google Scholar 

  • Fischer D, Pfitzner B, Schmid M, Simões-Araújo JL, Reis VM, Pereira W, Ormeño-Orrillo E, Hofmann A, Martinez-Romero E, Baldani JI, Hartmann A (2011) Molecular characterisation of the diazotrophic bacterial community in uninoculated and inoculated field-grown sugarcane (Saccharum sp.). Plant Soil doi:10.1007/s11104-011-0812-0

  • Franke-Whittle IH, Fegan M, Hayward C, Leonard G, Stackebrandt E, Sly LI (1999) Description of Gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug. Int J Syst Bacteriol 49:1681–1693

    Article  Google Scholar 

  • Franke-Whittle IH, O’Shea MG, Leonard GJ, Webb RI, Sly LI (2005) Investigation into the ability of Gluconacetobacter sacchari to live as an endophyte in sugarcane. Plant Soil 271:285–295

    Article  CAS  Google Scholar 

  • Furushita M, Shiba T, Maeda T, Yahata M, Kaneoka A, Takahashi Y, Trii K, Hasegawa T, Ohta M (2003) Similarity of tetracycline resistance genes applied isolated from fish farm bacteria to those from clinical isolates. Appl Environ Microb 69:5336–5342

    Article  CAS  Google Scholar 

  • Gyaneshwar P, James EK, Mathan N, Reddy PM, Reinhold-Hurek B, Ladha JK (2001) Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens. J Bacteriol 183:2634–2645

    Article  PubMed  CAS  Google Scholar 

  • Hartmann A, Bashan Y (2009) Ecology and application of Azospirillum and other plant growth-promoting bacteria (PGPB). Eur J Soil Biol 45:1–122

    Article  Google Scholar 

  • Hurek T, Wagner B, Reinhold-Hurek B (1997) Identification of N2-fixing plant- and fungus-associated Azoarcus species by PCR-based genomic fingerprints. Appl Environ Microb 63:4331–4339

    CAS  Google Scholar 

  • Islam MR, Madhaiyan M, Boruah HPD, Yim W, Lee G, Saravanan VS, Fu Q, Hu H, Sa T (2009) Characterization of plant growth-promoting traits of free-living diazotrophic bacteria and their inoculation effects on growth and nitrogen uptake of crop plants. J Microbiol Biotechn 19:1213–1222

    CAS  Google Scholar 

  • Jha B, Thakura MC, Gontia I, Albrecht V, Stoffels M, Schmid M, Hartmann A (2009) Isolation, partial identification and application of diazotrophic rhizobacteria from traditional Indian rice cultivars. Eur J Soil Biol 45:62–72

    Article  CAS  Google Scholar 

  • Juraeva D, George E, Davranov K, Ruppel S (2006) Detection and quantification of the nifH gene in shoot and root of cucumber plants. Can J Microbiol 52:731–739

    Article  PubMed  CAS  Google Scholar 

  • Kaschuk G, Hungria M, Andrade DS, Campo RJ (2006) Genetic diversity of rhizobia associated with common bean (Phaseolus vulgaris L.) grown under no-tillage and conventional systems in Southern Brazil. Appl Soil Ecol 32:210–220

    Article  Google Scholar 

  • Khan MS, Zaidi A, Wani PA, Ahemad M, Oves M (2009) Functional diversity among plant growth-promoting rhizobacteria. In: Khan MS, Zaidi A, Musarrat J (eds) Microbial strategies for crop improvement. Springer, Berlin, pp 105–132

    Chapter  Google Scholar 

  • Kirchhof G, Reis VM, Baldani JI, Eckert B, Döbereiner J, Hartmann A (1997a) Occurrence, physiological and molecular analysis of endophytic diazotrophic bacteria in gramineous energy plants. Plant Soil 104:45–55

    Article  Google Scholar 

  • Kirchhof G, Schloter M, Abmus B, Hartmann A (1997b) Molecular microbial ecology approaches applied to diazotrophs associated with non-legumes. Soil Biol Biochem 29:853–862

    Article  CAS  Google Scholar 

  • Kirchhof G, Eckert B, Stoffels M, Baldani JI, Reis VM, Hartmann A (2001) Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4-fiber plants. Int J Syst Evol Microbiol 51:157–168

    PubMed  CAS  Google Scholar 

  • Lerner A, Valverde A, Castro-Sowinski S, Lerner H, Okon Y, Burdman S (2010) Phenotypic variation in Azospirillum brasilense exposed to starvation. Environ Microbiol Rep 2:1758–2229

    Article  Google Scholar 

  • Mehta S, Nautiyal CS (2001) An efficient method for qualitative screening of phosphate-solubilizing bacteria. Curr Microbiol 43:51–56

    Article  PubMed  CAS  Google Scholar 

  • Moore FP, Barac T, Borremans B, Oeyen L, Vangronsveld J, van der Lelie D, Campbell D, Moore ERB (2006) Endophytic bacterial diversity in poplar trees growing on a BTEX-contaminated site: the characterisation of isolates with potential to enhance phytoremediation. Syst Appl Microbiol 29:539–556

    Article  PubMed  CAS  Google Scholar 

  • Morais RF, Souza BJ, Leite JM, Soares LH, Alves BJR, Boddey RM, Urquiaga S (2009) Elephant grass genotypes for bioenergy production by direct biomass combustion. Pesq Agropec Bras 44:133–140

    Google Scholar 

  • Morais RF, Quesada DM, Reis VM, Urquiaga S, Alves BJR, Boddey RM (2011) Contribution of biological nitrogen fixation to Elephant grass (Pennisetum purpureum Schum.). Plant Soil. doi:10.1007/s11104-011-0944-2

  • Nayak BS, Badgley B, Harwood VJ (2011) Comparison of genotypic and phylogenetic relationships of environmental Enterococcus isolates by BOX-PCR typing and 16S rRNA gene sequencing. Appl Environ Microbiol 77:5050–5055

    Article  PubMed  CAS  Google Scholar 

  • Oliveira ALM, Urquiaga S, Döbereiner J, Baldani JI (2002) The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants. Plant Soil 242:205–215

    Article  CAS  Google Scholar 

  • Pariona-Llanos R, Ibañez De Santi Ferrara Felipe F, Soto Gonzales HH, Barbosa HR (2010) Influence of organic fertilization on the number of cultivable diazotrophic endophytic bacteria isolated from sugarcane. Eur J Soil Biol 46:387–393

    Article  Google Scholar 

  • Pedraza RO, RamíRez-Mata A, Xiqui M, Baca BE (2004) Aromatic amino acid aminotransferase activity and indole-3-acetic acid production by associative nitrogen-fixing bacteria. FEMS Microbiol Lett 233:15–21

    Article  PubMed  CAS  Google Scholar 

  • Peng G, Zhang W, Luo H, Xie H, Lai W, Tan Z (2009) Enterobacter oryzae sp. nov., a nitrogen-fixing bacterium isolated from the wild rice species Oryza latifolia. Int J Syst Evol Microbiol 59:1650–1655

    PubMed  CAS  Google Scholar 

  • Perin L, Martinez-Aguilar L, Paredes Valdez G, Baldani JI, Estrada-De Los Santos P, Reis VM, Caballero-Mellado J (2006) Burkholderia silvatlantica sp. nov., a diazotrophic bacterium associated with sugarcane and maize. Int J Syst Evol Microbiol 56:1931–1937

    Article  PubMed  CAS  Google Scholar 

  • Poly F, Monrozier LJ, Bally R (2001) Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil. Res Microbiol 152:95–103

    Article  PubMed  CAS  Google Scholar 

  • Radwan TEE, Mohamed ZK, Reis VM (2002) Production of indole-3-acetic acid by different strains of Azospirillum and Herbaspirillum spp. Symbiosis 32:39–54

    CAS  Google Scholar 

  • Raymond J, Siefert JL, Staples CR, Blankenship RE (2004) The natural history of nitrogen fixation. Mol Biol Evol 21:541–554

    Article  PubMed  CAS  Google Scholar 

  • Reis VM, dos Reis FB Jr, Sales JF, Schloter M (2000) Characterisation of different polyclonal antisera to quantify Herbaspirillum spp. in Elephant grass (Pennisetum purpureum Schum). Symbiosis 29:139–150

    Google Scholar 

  • Reis VM, Reis Junior FB, Quesada DM, Oliveira OCA, Alves BJR, Urquiaga S, Boddey RM (2001) Biological nitrogen fixation associated with tropical pasture grasses. Aust J Plant Physiol 28:837–844

    Google Scholar 

  • Reis VM, Estrada-De Los Santos P, Tenorio-Salgado S, Vogel J, Stoffels M, Guyon S, Mavingui P, Baldani VLD, Schmid M, Baldani JI, Balandreau J, Hartmann A, Caballero-Mellado J (2004) Burkholderia tropica sp. nov., a novel nitrogen-fixing, plant-associated bacterium. Int J Syst Evol Microbiol 54:2155–2162

    Article  PubMed  CAS  Google Scholar 

  • Rodrigues E, Rodrigues LS, Oliveira ALM, Baldani VLD, Teixeira KRS, Urquiaga S, Reis VM (2008) Azospirillum amazonense inoculation: effects on growth, yield and N2 fixation of rice (Oryza sativa L.). Plant Soil 302:249–261

    Article  CAS  Google Scholar 

  • Rodríguez H, Fraga R (1999) Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol Adv 17:319–339

    Article  PubMed  Google Scholar 

  • Roesch LFW, Olivares FL, Passaglia LMP, Selbach PA, Sa ELS, Camargo FAO (2006) Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen supply. World J Microb Biot 22:967–974

    Article  Google Scholar 

  • Roy BD, Deb B, Sharma GD (2010) Role of acetic acid bacteria in biological N2 fixation- a review. Biofrontiers 1:47–57

    Google Scholar 

  • Samson R, Mani S, Boddey R, Sokhansanj S, Quesada D, Urquiaga S, Reis V, Ho Lem C (2005) The potential of C4perennial grasses for developing a global bioheat industry. Crit Rev Plant Sci 24:461–495

    Article  Google Scholar 

  • Sarwar M, Kremer RJ (1995) Enhanced suppression of plant growth through production of L-tryptophan-derived compounds by deleterious rhizobacteria. Plant Soil 172:261–269

    Article  CAS  Google Scholar 

  • Sashidhar B, Podile AR (2010) Mineral phosphate solubilization by rhizosphere bacteria and scope for manipulation of the direct oxidation pathway glucose dehydrogenase. J Appl Microbiol 109:1–12

    PubMed  CAS  Google Scholar 

  • Spaepen S, Vanderleyden J, Okon Y (2009) Plant growth-promoting actions of rhizobacteria. Adv Bot Res 7:283–320

    Article  Google Scholar 

  • Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T, Vangronsveld J, Van Der Lelie D (2009) Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl Environ Microb 75:748–757

    Article  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  PubMed  CAS  Google Scholar 

  • Vassilev N, Vassileva M (2003) Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes. Appl Microbiol Biot 61:435–440

    CAS  Google Scholar 

  • Versalovic J, Schneider M, De Bruijn FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Meth Mol Cell Biol 5:25–40

    CAS  Google Scholar 

  • Videira SS, Araújo JLS, Rodrigues LS, Baldani VLD, Baldani JI (2009) Occurrence and diversity of nitrogen-fixing Sphingomonas bacteria associated with rice plants grown in Brazil. FEMS Microbiol Lett 293:11–19

    Article  PubMed  CAS  Google Scholar 

  • Wang RF, Cao WW, Cerniglia CE (1996) Phylogenetic analysis of Fusobacterium prausnitzii based upon the 16S rRNA gene sequence and PCR confirmation. Int J Syst Bacteriol 46:341–343

    Article  PubMed  CAS  Google Scholar 

  • Xin G, Zhang GY, Kang JW, Staley JT, Doty SL (2009) A diazotrophic, indole-3-acetic acid-producing endophyte from wild cottonwood. Biol Fertil Soils 45:669–674

    Article  CAS  Google Scholar 

  • Yamada Y, Hoshino K, Ishikawa T (1997) The phylogeny of acetic acid bacteria based on the partial sequences of 16 s ribosomal RNA: the elevation of the subgenus Gluconoacetobacterium to generic level. Biosci Biotech Bioch 61:1244–1251

    Article  CAS  Google Scholar 

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Acknowledgment

The authors thank Embrapa Agrobiologia, the project CNPq/INCT-FBN, Faperj, CNPq (edital Universal) for financial support and CAPES and CNPq for the fellowship of the first and last two authors. The authors also thank the colleagues Robert M. Boddey and David Johnston-Monje for proof reading this manuscript.

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Authors and Affiliations

  1. Graduate student, Soil Sciences-UFRRJ/Embrapa Agrobiologia, Seropédica, RJ, Brazil

    Sandy Sampaio Videira & Rafael Fiusa de Morais

  2. Undergraduate student-UFRRJ/Embrapa Agrobiologia, Seropédica, RJ, Brazil

    Danilo Messias de Oliveira

  3. Research scientist-Embrapa Amapá, Rodovia Juscelino Kubitscheck, Km5, 2600, Macapá, AP, Brazil

    Wardsson Lustrino Borges

  4. Research Scientists-Embrapa Agrobiologia, BR 465, km 07, Seropédica, RJ, CEP 23890-000, Brazil

    Vera Lúcia Divan Baldani & José Ivo Baldani

  5. Embrapa Agrobiologia, BR 465, km07, 23890-000, Seropédica, Rio de Janeiro, Brazil

    José Ivo Baldani

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  1. Sandy Sampaio Videira
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  2. Danilo Messias de Oliveira
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  3. Rafael Fiusa de Morais
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Correspondence to José Ivo Baldani.

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Figure S1

Dendrogram obtained by cluster analysis of the BOX-PCR data for isolates from JNFb and NFb media. Similarities were calculated using the Dice coefficient and the data clustered using the unweighted pair group method of averages (UPGMA). Type strains are included (✰). Isolates indicated with the symbol “*” were used for partial sequencing of 16S rRNA while those indicated with “★” were used for total sequencing of 16S rRNA. (JPEG 137 kb)

Figure S2

Dendrogram obtained by cluster analysis of the BOX-PCR data for isolates from LGI medium. Similarities were calculated using the Dice coefficient and the data clustered using the unweighted pair group method of averages (UPGMA). Type strains are included (✰). Isolates indicated with the symbol “*” were used for partial sequencing of 16S rRNA while those indicated with “★” were used for total sequencing of 16S rRNA. (JPEG 69 kb)

Figure S3

Dendrogram obtained by cluster analysis of the BOX-PCR data for isolates from LGI-P medium. Similarities were calculated using the Dice coefficient and the data clustered using the unweighted pair group method of averages (UPGMA). Type strains are included (✰). Isolates indicated with the symbol “*” were used for partial sequencing of 16S rRNA while those indicated with “★” were used for total sequencing of 16S rRNA. (JPEG 106 kb)

Figure S4

Dendrogram obtained by cluster analysis of the BOX-PCR data for isolates from JMV medium. Similarities were calculated using the Dice coefficient and the data clustered using the unweighted pair group method of averages (UPGMA). Type strains are included (✰). Isolates indicated with the symbol “*” were used for partial sequencing of 16S rRNA while those indicated with “★” were used for total sequencing of 16S rRNA. (JPEG 146 kb)

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Videira, S.S., de Oliveira, D.M., de Morais, R.F. et al. Genetic diversity and plant growth promoting traits of diazotrophic bacteria isolated from two Pennisetum purpureum Schum. genotypes grown in the field. Plant Soil 356, 51–66 (2012). https://doi.org/10.1007/s11104-011-1082-6

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