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Phenotypic and genotypic characterization of endophytic bacteria associated with transgenic and non-transgenic soybean plants

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Abstract

Endophytic bacteria isolated from non-transgenic and transgenic Roundup Ready® glyphosate-resistant (GR) soybean plants were investigated to analyze the correspondence between phenotypic and genotypic characteristics and to determine whether or not the strains could be grouped based on the source of isolation in transgenic or non-transgenic plants, respectively. Most of the strains recovered from GR plants have shown the ability for plant growth promotion (PGP) by means of IAA production and inorganic phosphate solubilization, and 100% of the strains showed great motility (swarm or swim); in addition, 90% of the strains were able to metabolize the majority of carbon sources tested. GR soybean fields showed higher endophytes abundance than non-transgenic; however, analyzing the phylogenetic trees constructed using the partial 16SrRNA gene sequences, higher diversity was observed in non-transgenic soybean fields. Overall the majority of isolated endophytes could utilize multiple patterns of carbon sources and express resistance to antibiotics, while isolates varied widely in the PGP ability. The greater pattern and frequency of utilization of carbon sources and frequency and intensity of antibiotic resistance compared with PGP ability within the soybean endophytes community suggest that carbon sources metabolism and antibiotic resistance confer a greater relative fitness benefit than PGP ability. In conclusion, cluster analysis of the phenotypes and 16SrRNA gene sequences reveals lack of correspondence between the pattern of bacterial isolates and the transgenic character of plants, and the heterogeneity of clustering suggested that various adaptive processes, such as stress response, could have contributed to generate phenotypic variability to enhance endophytes overall fitness.

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References

  • Andreote FD, Mendes R, Dini-Andreote F, Rossetto PB, Labate CA, Pizzirani-Kleiner AA, van Elsas JD, Azevedo JL, Araújo WL (2008) Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development. Antonie Van Leeuwenhoek 93:415–424

    Article  CAS  PubMed  Google Scholar 

  • Andreote FD, Rocha UN, Araújo WL, Azevedo JL, Van Overbeek LS (2010) Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum). Antonie Van Leeuwenhoek 97:389–399

    Article  PubMed  PubMed Central  Google Scholar 

  • Anzai Y, Kudo Y, Oyaizu H (1997) The phylogeny of the genera Chryseomonas, Flavimonas, and Pseudomonas supports synonymy of these three genera. Int J Syst Bacteriol 47:249–251

    Article  CAS  PubMed  Google Scholar 

  • Assumpção LC (2008) Diversidade da comunidade bacteriana endofítica de sementes de soja e o seu potencial biotecnológico.p 93, Dissertation (Master in Agronomy)—Escola Superior de Agricultura Luiz de Queiroz, Piracicaba

  • Baines ALD, Xiao K, Kinkel LL (2007) Lack of correspondence between genetic and phenotypic groups amongst soil-borne streptomycetes. FEMS Microbiol Ecol 59:564–575

    Article  CAS  Google Scholar 

  • Barriuso J, Marín S, Mellado RP (2010) Effect of the herbicide glyphosate on glyphosate-tolerant maize rhizobacterial communities: a comparison with pre-emergency applied herbicide consisting of a combination of acetochlor and terbuthylazine. Environ Microbiol 12:1021–1030

    Article  CAS  PubMed  Google Scholar 

  • Böhm GMV, Rombaldi CV (2010) Genetic transformation and the use glyphosate on soil microbial, biological nitrogen fixation, quality and safety of genetically modified soybean. Cienc Rural 40:213–221

    Article  Google Scholar 

  • Bulgari D, Casati P, Brusetti L, Quaglino F, Brasca M, Daffonchio D, Bianco PA (2009) Endophytic bacterial diversity in Grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR. J Microbiol 47:393–401

    Article  CAS  PubMed  Google Scholar 

  • Busse MD, Ratcliff AW, Shestak CJ, Powers RF (2001) Glyphosate toxicity and the effects of long-term vegetation control on soil microbial communities. Soil Biol Biochem 33:1777–1789

    Article  CAS  Google Scholar 

  • Cerdeira AL, Duke SO (2006) The current status and environmental impacts of glyphosate-resistant crops: a review. J Environ Qual 35:1633–1658

    Article  CAS  PubMed  Google Scholar 

  • Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42:669–678

    Article  CAS  Google Scholar 

  • Davelos AL, Xiao K, Flor JM, Kinkel LL (2004) Genetic and phenotypic traits of streptomycetes used to characterize antibiotic activities of field-collected microbes. Can J Microbiol 50:79–89

    Article  CAS  PubMed  Google Scholar 

  • de Almeida Lopes KB, Carpentieri-Pipolo V, Oro TH, Stefani Pagliosa E, Degrassi G (2016) Culturable endophytic bacterial communities associated with field-grown soybean. J Appl Microbiol 120:740–755

    Article  CAS  PubMed  Google Scholar 

  • Di Giovanni GD, Watrud LS, Seidler RJ, Widmer F (1999) Comparison of parental and transgenic alfalfa rhizosphere bacterial communities using biolog GN metabolic fingerprinting and enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). Microb Ecol 37:129–139

    Article  PubMed  Google Scholar 

  • Döbereiner J, Baldani VL, Baldan JI (1995) Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas. EMBRAPA-SPI, Brasília, EMBRAPA-CNPAB, Itaguaí

  • Donegan KK, Palm CJ, Fieland VJ, Porteous LA, Ganio LM, Schaller DL, Bucao LQ, Seidler RJ (1995) Changes in levels, species and DNA fingerprints of soil microorganisms associated with cotton expressing the Bacillus thuringiensis var. kursmki endotoxin. Appl Soil Ecol 2:111–124

    Article  Google Scholar 

  • Duke SO, Lydon J, Koskinen WC, Moorman TB, Chaney RL, Hammerschmidt R (2012) Glyphosate effects on plant mineral nutrition, crop rhizosphere microbiota, and plant disease in glyphosate-resistant crops. J Agric Food Chem 60:10375–10397

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Egamberdiyeva D, Hoflich G (2003) Influence of growth- promoting bacteria on the growth of wheat in different soils and temperatures. Soil Biol Biochem 35:973–978

    Article  CAS  Google Scholar 

  • Embrapa (2006) Sistema Brasileiro de Classificação de Solos, 2nd edn. EMBRAPA-SPI, Rio de Janeiro, pp 306

  • Etesami H, Maheshwari DK (2018) Use of plant growth promoting rhizobacteria (PGPRs) with multiple plant growth promoting traits in stress agriculture: action mechanisms and future prospects. Ecotoxicol Environ Saf 156:225–246

    Article  CAS  PubMed  Google Scholar 

  • Etesami H, Mirseyed Hosseini H, Alikhani HA (2014) In planta selection of plant growth promoting endophytic bacteria for rice (Oryza sativa L.). J Soil Sci Plant Nutr 14:491–503

    CAS  Google Scholar 

  • Fischer RS, Berry A, Gaines CG, Jensen RA (1986) Comparative action of glyphosate as a trigger of energy drain in eubacteria. J Bacteriol 168:1147–1154

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fisher LM, Albery WJ, Knowles JR (1986) Energetics of proline racemase: tracer perturbation experiments using [14C] proline that measure the interconversion rate of the two forms of free enzyme. Biochem 25:2538–2542

    Article  CAS  Google Scholar 

  • Franz JE, Mao MK, Sikorski JA (1997) Glyphosate: a unique global herbicide. American Chemical Society, Washington, DC, p 653

    Google Scholar 

  • Freitas DB, Reis MP, Lima-Bittencourt CI, Costa PS, Assis PS, Chartone-Souza E, Nascimento AMA (2008) Genotypic and phenotypic diversity of Bacillus spp. isolated from steel plant waste. BMC Res Notes 92:1–11

    Google Scholar 

  • Gaiero JR, McCall CA, Thompson KA, Day NJ, Best AS, Dunfield KE (2013) Inside the root microbiome: bacterial root endophytes and plant growth promotion. Am J Bot 100:1738–1750

    Article  PubMed  Google Scholar 

  • Garipova SR (2014) Perspectives on using endophytic bacteria for the bioremediation of arable soils polluted by residual amounts of pesticides and xenobiotics. Biol Bull Rev 4:300–310

    Article  Google Scholar 

  • Germaine KJ, Liu X, Cabellos GG, Hogan JP, Ryan D, Dowling DN (2006) Bacterial entophyte-enhanced phytoremediation of the organochlorine herbicide 2,4-dichlorophenoxyacetic acid. FEMS Microbiol Ecol 57:302–310

    Article  CAS  PubMed  Google Scholar 

  • Glick BR, Stearns JC (2011) Making phytoremediation work better: maximizing a plant’s growth potential in the midst of adversity. Int J Phytoremediation 13:4–16

    Article  PubMed  Google Scholar 

  • Goes KCGP, Fisher MLC, Cattelan AJ, Nogueira MA, Carvalho CGPC, Oliveira ALM (2012) Biochemical and molecular characterization of high population density bacteria isolated from sunflower. J Microbiol Biotechnol 22:437–447

    Article  CAS  Google Scholar 

  • Guijarro KH, Aparicio V, De Gerónimo E, Castellote M, Figuerola EL, Costa JL, Erijman L (2018) Soil microbial communities and glyphosate decay in soils with different herbicide application history. Sci Total Environ 634:974–982

    Article  CAS  PubMed  Google Scholar 

  • Hardoim PH, van Overbeek LS, van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471

    Article  CAS  PubMed  Google Scholar 

  • Hungria M, Mendes IC, Nakatani AS, Reis-Junior FB, Morais JZ, Oliveira MCN, Fernandes MF (2014) Effects of the glyphosate-resistance gene and herbicides on soybean: field trials monitoring biological nitrogen fixation and yield. Field Crops Res 158:43–54

    Article  Google Scholar 

  • Jaworski EG (1972) Mode of action of N-phosphonomethylglycine: inhibition of aromatic amino acid biosynthesis. J Agr Food Chem 20:1195–1198

    Article  CAS  Google Scholar 

  • Kado CI, Heskett MG (1970) Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopatology 60:969–976

    Article  CAS  Google Scholar 

  • Kang SH, Hyun-Soo C, Hoon C, Choong-Min R, Jihyun FK, Seung-Hwan P (2007) Two bacterial entophytes eliciting both plant growth promotion and plant defense on pepper (Capsicum annuum L.). J Microbiol Biotechnol 17:96–103

    CAS  PubMed  Google Scholar 

  • Khalifa AYZ, Alsyeeh AM, Almalki AM, Saleh FA (2016) Characterization of the plant growth promotiong bacterium, Enterobacter cloacae MSR1, isolated from roots of non-nodulating Medicago sativa. Saudi J Biol Sci 23:79–86

    Article  CAS  PubMed  Google Scholar 

  • Kuklinsky-Sobral J, Araújo WL, Mendes R, Geraldi IO, Pizzirani-Kleiner AA, Azevedo JL (2004) Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion. Environ Microbiol 6:1244–1251

    Article  CAS  PubMed  Google Scholar 

  • Kuklinsky-Sobral J, Araújo WL, Mendes R, Pizzirani-Kleiner AA, Azevedo JL (2005) Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide. Plant Soil 273:91–99

    Article  CAS  Google Scholar 

  • Kumar D, Kumar L, Nagar S, Raina C, Parshad R, Gupta VK (2012) Screening, isolation and production of lipase/esterase producing Bacillus sp. strain DVL2 and its potential evaluation in esterification and resolution reactions. Arch Appl Sci Res 4:1763–1770

    CAS  Google Scholar 

  • Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175

    Google Scholar 

  • Liu Y, Feng Y, Cheng D, Xue J, Wakelin S, Li Z (2018) Dynamics of bacterial composition and the fate of antibiotic resistance genes and mobile genetic elements during the co-composting with gentamicin fermentation residue and lovastatin fermentation residue. Bioresour Technol 261:249–256

    Article  CAS  PubMed  Google Scholar 

  • Loper JE, Schroth MN (1986) Influence of bacterial sources of indole-2-acetic acid on root elongation of sugar beet. Phytopathology 76:386–389

    Article  CAS  Google Scholar 

  • Lottmann J, Berg G (2001) Phenotypic and genotypic characterization of antagonistic bacteria associated with roots of transgenic and non-transgenic potato plants. Microbiol Res 156:75–82

    Article  CAS  PubMed  Google Scholar 

  • Markova YuA, Romanenko AS, Dukhanina AV (2005) Isolation of bacteria of the family Enterobacteriaceae from plant tissue. Microbiology 74:663–666

    Article  CAS  PubMed  Google Scholar 

  • Miliute I, Buzaite O, Baniulis D, Stanys V (2015) Bacterial endophytes in agricultural crops and their role in stress tolerance: a review. Zemdirbyste-Agriculture 102:465–478

    Article  Google Scholar 

  • Murray TS, Kazmierczak BI (2006) FlhF is required for swimming and swarming in Pseudomonas aeruginosa. J Bacteriol 188:6995–7004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nautiyal CS (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170:265–270

    Article  CAS  PubMed  Google Scholar 

  • Oliveira RS, Dvoranen EC, Constantin J, Cavalieri SD, Franchini LHM, Rios FA, Blainski E (2008) Glyphosate influence on growth and nodulation of glyphosate resistant soybean cultivars. Planta Daninha 26:831–843

    Article  Google Scholar 

  • Padgette SR, Kolacz KH, Delannay X, Re DB, LaVallee BJ, Tinius CN, Rhodes WK, Otero YI, Barry GF, Eichholtz DA, Peschke VM, Nida DL, Taylor NB, Kishore GM (1995) Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci 35:1451–1461

    Article  CAS  Google Scholar 

  • Pline-Srnic W (2005) Technical performance of some commercial glyphosate-resistant crops. Pest Manag Sci 61:225–234

    Article  CAS  PubMed  Google Scholar 

  • Reddy KN, Duke SO (2014) Soybean mineral composition and glyphosate use. In: Preedy VR (ed) Processing and impact on active food components in food. Elsevier Inc, London, pp 369–376

    Google Scholar 

  • Rösch C, Bothe H (2005) Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes. Appl Environ Microbiol 71:2026–2035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sagova-Mareckova M, Daniel O, Omelka M, Kristufek V, Divis J, Kopecky J (2015) Determination of factors associated with natural soil suppressivity to potato common scab. PLoS One 10:1–13

    Article  CAS  Google Scholar 

  • Schulz B, Boyle C (2006) What are endophytes? In: Schulz B, Boyle CJ, Sieber TN (eds) Microbial root endophytes, vol 9. Springer, Berlin, pp 1–13

    Chapter  Google Scholar 

  • Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:46–56

    Article  Google Scholar 

  • Siciliano SD, Germida JJ (1999) Taxonomic diversity of bacteria associated with the roots of field-grown transgenic Brassica napus cv. Quest, compared to the non-transgenic B. napus cv. Excel and B. rapa cv. Parkland. FEMS Microbiol Ecol 29:263–272

    Article  CAS  Google Scholar 

  • Siciliano SD, Fortin N, Mihoc A, Wisse G, Labelle S, Beaumier D, Ouellette D, Roy R, Whyte LG, Banks MK, Schwab P, Lee K, Greer CH (2001) Selection of specific endophytic bacterial genotypes by plants in response to soil contamination. Appl Environ Microbiol 67:2469–2475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sikorski J, Pukall R, Stackebrandt E (2008) Carbon source utilization patterns of Bacillus simplex ecotypes do not reflect their adaptation to ecologically divergent slopes in ‘Evolution Canyon’, Israel. FEMS Microbiol Ecol 66:38–44

    Article  CAS  PubMed  Google Scholar 

  • Sturz AV, Christie BR, Nowak J (2000) Bacterial endophytes: potential role in developing sustainable systems of crop production. Crit Rev Plant Sci 19:1–30

    Article  Google Scholar 

  • Torsvik V, Salte K, Sorheim R, Goksoyr J (1990) Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria. Appl Environ Microbiol 56:776–781

    CAS  PubMed  PubMed Central  Google Scholar 

  • Vela AI, Vázquez J, Gibello A, Blanco MM, Moreno MA, LiéBana P, Albendea C, Alcala B, Mendez A, Dominguez L, Fernández-Garayzábal JF (2000) Phenotypic and genetic characterization of Lactococcus garvieae isolated in Spain from Lactococcosis outbreaks and comparison with isolates of other countries and sources. J Clin Microbiol 38:3791–3795

    CAS  PubMed  PubMed Central  Google Scholar 

  • Vigani G, Rolli E, Marasco R, Dell’Orto M, Michoud G, Soussi A, Raddadi N, Borin S, Sorlini C, Zocchi G, Daffonchio D (2018) Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H + ‐pumping pyrophosphatase in pepper plants. Environ Microbiol. https://doi.org/10.1111/1462-2920.14272

  • Wen CM, Tseng CS, Cheng CY, Li YK (2002) Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2. Biotechnol Appl Biochem 35:213–219

    Article  CAS  PubMed  Google Scholar 

  • Xing D, Ren N, Wang A, Li Q, Feng Y, Ma F (2008) Continuous hydrogen production of auto-aggregative Ethanoligenens harbinense YUAN-3 under non-sterile condition. Int J Hydrogen Energy 33:1489–1495

    Article  CAS  Google Scholar 

  • Zablotowicz RM, Reddy KN (2004) Impact of glyphosate on the Bradyrhizobium japonicum symbiosis with glyphosate-resistant transgenic soybean: a minireview. J Environ Qual 33:825–831

    Article  CAS  PubMed  Google Scholar 

  • Zhang J, Tang M, Viikari L (2012) Xylans inhibit enzymatic hydrolysis of lignocellulosic materials by cellulases. Bioresour Technol 121:8–12

    Article  CAS  PubMed  Google Scholar 

  • Zinniel DK, Lambrecht P, Harris NB, Feng Z, Kuczmarski D, Higley P, Ishimaru CA, Arunakumari A, Barletta RG, Vidaver AK (2002) Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl Environ Microbiol 68:2198–2208

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zlosnik JE, Hird TJ, Frankel MC, Moreira LM, Henry DA, Speert DP (2008) Differential mucoid exopolysaccharide production by members of the Burkholderia cepacia complex. J Clin Microbiol 46:1470–1473

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This research was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Program Science Without Borders, Grant 019/2012, and by Fundação Araucária (Brazil), Grant 23265/2011. We thank ICGEB (International Centre For Genetic Engineering and Biotechnology, Trieste, Italy) for the SMART Fellowship to Karla Bianca de Almeida Lopes. We also thank Jennifer Aparecida Schnitzer for laboratory assistance.

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  1. Embrapa Trigo, Rodovia BR-285, Km 294, PO Box 3081, Passo Fundo, RS, 99050-970, Brazil

    Valeria Carpentieri-Pipolo

  2. Agronomy Department, Londrina State University, Rodovia Celso Garcia Cid, Pr 445 km 380, PO Box 10.011, Londrina, PR, 86057-970, Brazil

    Karla Bianca de Almeida Lopes

  3. IBioBA-ICGEB, International Centre for Genetic Engineering and Biotechnology, Polo Cientifico Tecnologico, Godoy Cruz 2390, C1425FQD, Buenos Aires, Argentina

    Giuliano Degrassi

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  1. Valeria Carpentieri-Pipolo
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Communicated by Erko Stackebrandt.

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Carpentieri-Pipolo, V., de Almeida Lopes, K.B. & Degrassi, G. Phenotypic and genotypic characterization of endophytic bacteria associated with transgenic and non-transgenic soybean plants. Arch Microbiol 201, 1029–1045 (2019). https://doi.org/10.1007/s00203-019-01672-6

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