Environmental Science and Pollution Research

, Volume 23, Issue 13, pp 13255–13267 | Cite as

Diversity of endophytic Pseudomonas in Halimione portulacoides from metal(loid)-polluted salt marshes

  • Jaqueline Rocha
  • Marta Tacão
  • Cátia Fidalgo
  • Artur Alves
  • Isabel Henriques
Research Article


Phytoremediation assisted by bacteria is seen as a promising alternative to reduce metal contamination in the environment. The main goal of this study was to characterize endophytic Pseudomonas isolated from Halimione portulacoides, a metal-accumulator plant, in salt marshes contaminated with metal(loid)s. Phylogenetic analysis based on 16S rRNA and gyrB genes showed that isolates affiliated with P. sabulinigri (n = 16), P. koreensis (n = 10), P. simiae (n = 5), P. seleniipraecipitans (n = 2), P. guineae (n = 2), P. migulae (n = 1), P. fragi (n = 1), P. xanthomarina (n = 1), and Pseudomonas sp. (n = 1). Most of these species have never been described as endophytic. The majority of the isolates were resistant to three or more metal(loid)s. Antibiotic resistance was frequent among the isolates but most likely related to species-intrinsic features. Common acquired antibiotic resistance genes and integrons were not detected. Plasmids were detected in 43.6 % of the isolates. Isolates that affiliated with different species shared the same plasmid profile but attempts to transfer metal resistance to receptor strains were not successful. Phosphate solubilization and IAA production were the most prevalent plant growth promoting traits, and 20 % of the isolates showed activity against phytopathogenic bacteria. Most isolates produced four or more extracellular enzymes. Preliminary results showed that two selected isolates promote Arabidopsis thaliana root elongation. Results highlight the diversity of endophytic Pseudomonas in H. portulacoides from contaminated sites and their potential to assist phytoremediation by acting as plant growth promoters and as environmental detoxifiers.


Pseudomonas Halimione portulacoides Endophytic Phytoremediation Plant growth promoters Metals 



This work was supported by European Funds (FEDER) through COMPETE and by National Funds through the Portuguese Foundation for Science and Technology (FCT) within project PhytoMarsh (PTDC/AAC-651 AMB/118873/2010-FCOMP-01-0124-FEDER-019328). Authors also acknowledge FCT financing to CESAM (UID/AMB/50017/2013) and iBiMED (UID/BIM/04501/2013), Artur Alves (FCT Investigator Programme–IF/00835/2013), Isabel Henriques (FCT Investigator Programme–IF/00492/2013) and Cátia Fidalgo (PhD grant SFRH/BD/85423/2012). The authors wish to thank Sofia Pereira and Paula Castro (Universidade Católica Portuguesa, Portugal) for providing positive controls for plant-growth promotion traits screening, Kornelia Smalla (Julius Kuhn Institut, Germany) for the phytopathogenic strains used in antimicrobial activity assays.

Supplementary material

11356_2016_6483_MOESM1_ESM.pdf (223 kb)
ESM 1 (PDF 222 kb)


  1. Aguilar-Barajas E, Ramírez-Díaz MI, Riveros-Rosas H, Cervantes C (2010) Heavy metal resistance in Pseudomonads. In: Ramos J, Filloux A (Eds.). Pseudomonas, Volume 6, Springer, pp. 255–282Google Scholar
  2. Ali S, Charles TC, Glick BR (2014) Amelioration of high salinity stress damage by plant growth-promoting bacterial endophytes that contain ACC deaminase. Plant Physiol Biochem 80:160–167. doi: 10.1016/j.plaphy.2014.04.003 CrossRefGoogle Scholar
  3. Almeida CMR, Dias AC, Mucha AP, Bordalo AA, Vasconcelos MTSD (2009) Influence of surfactants on the Cu phytoremediation potential of a salt marsh plant. Chemosphere 75:135–140. doi: 10.1016/j.chemosphere.2008.12.037 CrossRefGoogle Scholar
  4. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. doi: 10.1016/S0022-2836(05)80360-2 CrossRefGoogle Scholar
  5. Ando S, Goto M, Meunchang S, Thongra-ar P, Fujiwara T, Hayashi H, Yoneyama T (2005) Detection of nifH sequences in sugarcane (Saccharum officinarum L.) and pineapple (Ananas comosus [L.] Merr.). Soil Sci Plant Nutr 51:303–308. doi: 10.1111/j.1747-0765.2005.tb00034.x CrossRefGoogle Scholar
  6. Anjum NA, Ahmad I, Válega M, Pacheco M, Figueira E, Duarte AC, Pereira E (2011) Impact of seasonal fluctuations on the sediment-mercury, its accumulation and partioning in Halimione portulacoides and Juncus maritimus collected from Ria de Aveiro coastal lagoon (Portugal). Water Air Soil Pollut 222:1–15. doi: 10.1007/s11270-001-0799-4 CrossRefGoogle Scholar
  7. Anyakora C, Ehianeta T, Umukoro O (2013) Heavy metal levels in soil samples from highly industrialized Lagos environment. Afr J Environ Sci Technol 7:917–924. doi: 10.5897/AJEST2013.1543 Google Scholar
  8. Babu AG, Shea PJ, Sudhakar D, Jung I, Oh B (2015) Potential use of Pseudomonas koreensis AGB-1 in association with Miscanthus sinensis to remediate heavy metal(loid)-contaminated mining site soil. J Environ Manag 151:160–166. doi: 10.1016/j.jenvman.2014.12.045 CrossRefGoogle Scholar
  9. Baker-Austin C, Wright MS, Stepanauskas R, McArthur JV (2006) Co-selection of antibiotic and metal resistance. Trends Microbiol 14:176–182. doi: 10.1016/j.tim.2006.02.006 CrossRefGoogle Scholar
  10. Cambrollé J, Mancilla-Leytón JM, Muñoz-Vallés S, Luque T, Figueroa MT (2012a) Zinc tolerance and accumulation in the salt-marsh shrub Halimione portulacoides. Chemosphere 86:867–874. doi: 10.1016/j.chemosphere.2011.10.039 CrossRefGoogle Scholar
  11. Cambrollé J, Mancilla-Leytón JM, Muñoz-Vallés S, Luque T, Figueroa ME (2012b) Tolerance and accumulation of copper in the salt-marsh shrub Halimione portulacoides. Mar Pollut Bull 64:721–728. doi: 10.1016/j.marpolbul.2012.02.002 CrossRefGoogle Scholar
  12. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ (2005) Identification of plasmids by PCR-based replicon typing. J Microbiol methods 63:219–228. doi: 10.1016/j.mimet.2005.03.018 CrossRefGoogle Scholar
  13. Carvalho PN, Basto MCP, Silva MFGM, Machado A, Bordalo AA, Vasconcelos MTSD (2010) Ability of salt marsh plants for TBT remediation in sediments. Environ Sci Pollut R 17:1279–1286. doi: 10.1007/s11356-010-0307-1 CrossRefGoogle Scholar
  14. Chaturvedi S, Chandra R, Rai V (2006) Isolation and characterization of Phragmites australis (L.) rhizosphere bacteria from contaminated site for bioremediation of colored distillery effluent. Ecol Eng 27:202–207. doi: 10.1016/j.ecoleng.2006.02.008 CrossRefGoogle Scholar
  15. Chauhan H, Bagyaraj DJ, Selvakumar G, Sundaram SP (2015) Novel plant growth promoting rhizobacteria—prospects and potential. Appl Soil Ecol 95:38–53. doi: 10.1016/j.apsoil.2015.05.011 CrossRefGoogle Scholar
  16. CLSI (2012) Performance standard for antimicrobial susceptibility testing—document approved standard M100-S22. CLSI, WayneGoogle Scholar
  17. 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. doi: 10.1016/j.soilbio.2009.11.024 CrossRefGoogle Scholar
  18. Costa C, Jesus-Rydin C (2001) Site investigation on heavy metals contaminated ground in Estarreja—Portugal. Eng Geol 60:39–47. doi: 10.1016/S0013-7952(00)00087-9 CrossRefGoogle Scholar
  19. Couto MNPFS, Basto MCP, Vasconcelos MTSD (2011) Suitability of different salt marsh plants for petroleum hydrocarbons remediation. Chemosphere 84:1052–1057. doi: 10.1016/j.chemosphere.2011.04.069 CrossRefGoogle Scholar
  20. Deredjian A, Colinon C, Brothier E, Favre-Bonté S, Cournoyer B, Nazaret S (2011) Antibiotic and metal resistance among hospital and outdoor strains of Pseudomonas aeruginosa. Res Microbiol 162:689–700. doi: 10.1016/j.resmic.2011.06.007 CrossRefGoogle Scholar
  21. Duarte B, Delgado M, Caçador I (2007) The role of citric acid in cadmium and nickel uptake and translocation, in Halimione portulacoides. Chemosphere 69:836–840. doi: 10.1016/j.chemosphere.2007.05.007 CrossRefGoogle Scholar
  22. Dworkin M, Foster JW (1958) Experiments with some microorganisms which utilize ethane and hydrogen. J Bacteriol 75:592–603Google Scholar
  23. Fidalgo C, Henriques I, Rocha J, Tacao M, Alves A (2016) Culturable endophytic bacteria from the salt marsh plant Halimione portulacoides: phylogenetic diversity, functional characterization and influence of metal(loid) contamination. Environ Sci Pollut R. doi: 10.1007/s11356-016-6208-1
  24. Gaby JC, Buckley DH (2012) A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase. PLoS ONE 7, e42149. doi: 10.1371/journal.pone.0042149 CrossRefGoogle Scholar
  25. Ganeshan G, Kumar AM (2005) Pseudomonas fluorescens, a potential bacterial antagonist to control plant diseases. J Plant Interact 1:123–134. doi: 10.1080/17429140600907043 CrossRefGoogle Scholar
  26. Gordon SA, Weber RP (1951) Colorimetric estimation of indoleacetic acid. Plant Physiol 26:192–195. doi: 10.1016/0003-2697(76)90514-5 CrossRefGoogle Scholar
  27. Kim K, Roh SW, Chang H, Nam Y, Yoon J, Jeon CO, Oh H, Bae J (2009) Pseudomonas sabulinigri sp. nov., isolated from black beach sand. Int J Syst Evol Microbiol 59:38–41. doi: 10.1099/lijs0.65866-0 CrossRefGoogle Scholar
  28. Kim O, Cho Y, Lee K, Yoon S, Kim M, Na H, Park S, Jeon Y, Lee J, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721. doi: 10.1099/ijs.0.038075-0 CrossRefGoogle Scholar
  29. Livermore DM (2001) Of Pseudomonas, porins, pumps and carbapenems. J Antimicrob Chemother 47(3):247–250. doi: 10.1093/jac/47.3.247 CrossRefGoogle Scholar
  30. Long HH, Schmidt DD, Baldwin IT (2008) Native bacterial endophytes promote host growth in a species-specific manner; phytohormone manipulations do not result in common growth responses. PLoS One 3, e2702. doi: 10.1371/journal.pone.0002702 CrossRefGoogle Scholar
  31. Lopez-Velasco G, Tydings HA, Boyer RR, Falkinham JO, Ponder MA (2012) Characterization of interactions between Escherichia coli O157:H7 with epiphytic bacteria in vitro and on spinach leaf surfaces. Int J Food Microbiol 153:351–357. doi: 10.1016/j.ijfoodmicro.2011.11.026 CrossRefGoogle Scholar
  32. Ma Y, Prasad MNV, Rajkumar M, Freitas H (2011) Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils. Biotechnol Adv 29:248–258. doi: 10.1016/j.biotechadv.2010.12.001 CrossRefGoogle Scholar
  33. Malik A, Aleem A (2011) Incidence of metal and antibiotic resistance in Pseudomonas spp. from the river water, agricultural soil irrigated with wastewater and ground water. Environ Monit Assess 178:293–308. doi: 10.1007/s10661-010-1690-2 CrossRefGoogle Scholar
  34. Malik A, Jaiswal R (2000) Metal resistance in Pseudomonas strains isolated from soil treated with industrial wastewater. World J microbiol Biot 16:177–182. doi: 10.1023/A:1008905902282 CrossRefGoogle Scholar
  35. Martins V (2011) Comunidade bacteriana endofítica cultivável de Halimione portulacoides. Dissertation, University of Aveiro.
  36. Mesaros N, Nordmann P, Plésia P, Roussel-Delvallez M, Van Eldere J, Glupczynski Y, Van Laethem Y, Jacobs F, Lebecque P, Malfroot A, Tulkens PM, Van Bambeke F (2007) Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect 13(6):560–578. doi: 10.1111/j.1469-0691.2007.01681.x CrossRefGoogle Scholar
  37. Mucha AP, Almeida CMR, Magalhães CM, Vasconcelos MTSD, Bordalo AA (2011) Salt marsh plant-microorganism interaction in the presence of mixed contamination. Int Biodeter Biodegr 65:326–333. doi: 10.1016/j.ibiod.2010.12.005 CrossRefGoogle Scholar
  38. Nautiyal CS (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170:265–270. doi: 10.1016/S0378-1097(98)00555-2 CrossRefGoogle Scholar
  39. Nies DH (1999) Microbial heavy-metal resistance. Appl Microbiol Biot 51:730–750CrossRefGoogle Scholar
  40. Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 118:10–15. doi: 10.1034/j.1399-3054.2003.00086.x CrossRefGoogle Scholar
  41. Pereira SIA, Barbosa L, Castro PML (2015) Rhizobacteria isolated from a metal-polluted area enhance plant growth in zinc and cadmium-contaminated soil. Int J Environ Sci Technol 12:2127–2142. doi: 10.1007/s13762-014-0614-z CrossRefGoogle Scholar
  42. Pereira SIA, Monteiro C, Vega AL, Castro PML (2016) Endophytic culturable bacteria colonizing Lavandula dentata L. plants: isolation, characterization and evaluation of their plant growth-promoting activities. Ecol Eng 87:91–97. doi: 10.1016/j.ecoleng.2015.11.033 CrossRefGoogle Scholar
  43. Pérez-Miranda S, Fernández FJ (2013) Siderophores: what are they, and how are they detected? In: Amaya MG, Pacheco SV (eds) The struggle for iron: pathogen vs host. Cinvestav, MexicoGoogle Scholar
  44. Pérez-Miranda S, Cabirol N, George-Téllez R, Zamudio-Rivera LS, Fernández FJ (2007) O-CAS, a fast and universal method for siderophore detection. J Microbiol methods 70:127–131. doi: 10.1016/j.mimet.2007.03.023 CrossRefGoogle Scholar
  45. Petatán-Sagahón I, Anducho-Reyes MA, Silva-Rojas HV, Arana-Cuenca A, Tellez-Jurado A, Cárdenas-Álvarez IO, Mercado-Flores Y (2011) Isolation of bacteria with antifungal activity against the phytopathogenic fungi Stenocarpella maydis and Stenocarpella macrospora. Int J Mol Sci 12:5522–5537. doi: 10.3390/ijms12095522 CrossRefGoogle Scholar
  46. Poole K (2011) Pseudomonas aeruginosa: resistance to the max. Front Microbiol 2:1–13. doi: 10.3389/fmicb.2011.00065 CrossRefGoogle Scholar
  47. Prado S, Montes J, Romalde JL, Barja JL (2009) Inhibitory activity of Phaeobacter strains against aquaculture pathogenic bacteria. Int Microbiol 12:107–114. doi: 10.2436/20.1501.01.87 Google Scholar
  48. Preston GM (2004) Plant perceptions of plant growth-promoting Pseudomonas. Philos T R Soc Lon B 359:907–918. doi: 10.1098/rstb.2003.1384 CrossRefGoogle Scholar
  49. Proença DN, Francisco R, Santos CV, Lopes A, Fonseca L, Abrantes IMO, Morais PV (2010) Diversity of bacteria associated with Bursaphelenchus xylophilus and other nematodes isolated from Pinus pinaster trees with pine wilt disease. PLoS One 5(12), e15191. doi: 10.1371/journal.pone.0015191 CrossRefGoogle Scholar
  50. Rajkumar M, Ae N, Freitas H (2009) Endophytic bacteria and their potential to enhance heavy metal phytoextraction. Chemosphere 77:153–160. doi: 10.1016/j.chemosphere.2009.06.047 CrossRefGoogle Scholar
  51. Reetha AK, Pavani SL, Mohan S (2014) Hydrogen cyanide production ability by bacterial antagonist and their antibiotics inhibition potential on Macrophomina phaseolina (Tassi.) Goid. Int J Curr Microbiol Appl Sci 3(5):172–178Google Scholar
  52. R Core Team (2014) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL
  53. Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent developmentsand applications. FEMS Microbiol Lett 278:1–9. doi: 10.111/j.1574-6968.2007.00918.x CrossRefGoogle Scholar
  54. Shamim S, Rehman A (2013) Antioxidative enzyme profiling and biosorption ability of Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 under cadmium stress. J Basic Microbiol 55:374–381. doi: 10.1002/jobm.201300038 CrossRefGoogle Scholar
  55. Shi CL, Park HB, Lee JS, Ryu S, Ryu CM (2010) Inhibition of primary roots and stimulation of lateral root development in Arabidopsis thaliana by the rhizobacterium Serratia marcescens 90–166 is through both auxin-dependent and -independent signaling pathways. Mol Cells 29:251–258. doi: 10.1007/s10059-010-0032-0 CrossRefGoogle Scholar
  56. Singh P, Cameotra SS (2004) Enhancement of metal bioremediation by use of microbial surfactants. Biochem Biophys Res Commun 319:291–297. doi: 10.1016/j.bbrc.2004.04.155 CrossRefGoogle Scholar
  57. Sousa AI, Caçador I, Lillebø AI, Pardal MA (2008) Heavy metal accumulation in Halimione portulacoides: intra- and extra-cellular metal binding sites. Chemosphere 70:850–857. doi: 10.1016/j.chemosphere.2007.07.012 CrossRefGoogle Scholar
  58. Spiers AJ, Buckling A, Rainey PB (2000) The causes of Pseudomonas diversity. Microbiology 146:2345–2350CrossRefGoogle Scholar
  59. Stout L, Nüsslein K (2010) Biotechnological potential of aquatic plant-microbe interactions. Curr Opin Biotechnol 21:339–345. doi: 10.1016/j.copbio.2010.04.004 CrossRefGoogle Scholar
  60. Tacão M, Moura A, Correia A, Henriques I (2014) Co-resistance to different classes of antibiotics among ESBL-producers from aquatic systems. Water Res 48:100–107. doi: 10.1016/j.watres.2013.09.021 CrossRefGoogle Scholar
  61. Tacão M, Correia A, Henriques IS (2015) Low prevalence of carbapenem-resistant bacteria in river water: resistance is mostly related to intrinsic mechanisms. Microb Drug Resist 21:497–506. doi: 10.1089/mdr.2015.0072 CrossRefGoogle Scholar
  62. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. doi: 10.1093/molbev/mst197 CrossRefGoogle Scholar
  63. Tarkowski P, Vereecke D (2014) Threats and opportunities of plant pathogenic bacteria. Biotechnol Adv 32:215–229. doi: 10.1016/j.biotechadv.2013.11.001 CrossRefGoogle Scholar
  64. Toribio J, Escalante AE, Caballero-Mellado J, González-González A, Zavala S, Souza V, Soberón-Chávez G (2011) Characterization of a novel biosurfactant producing Pseudomonas koreensis lineage that is endemic to Cuatro Ciénegas Basin. Syst Appl Microbiol 34:531–535. doi: 10.1016/j.syapm.2011.01.007 CrossRefGoogle Scholar
  65. Ullah A, Heng S, Munis MFH, Fhad S, Yang X (2015) Phytoremediation of heavy metals assisted by plant growth promoting (PGP) bacteria: a review. Environ Exp Bot 117:28–40. doi: 10.1016/j.envexpbot.2015.05.001 CrossRefGoogle Scholar
  66. Válega M, Lillebø AI, Pereira ME, Caçador I, Duarte AC, Pardal MA (2008a) Mercury in salt marshes ecosystems: Halimione portulacoides as biomonitor. Chemosphere 73:1224–1229. doi: 10.1016/j.chemosphere.2008.07.053 CrossRefGoogle Scholar
  67. Válega M, Lillebø AI, Pereira ME, Duarte AC, Pardal MA (2008b) Long-term effects of mercury in a salt marsh: hysteresis in the distribution of vegetation following recovery from contamination. Chemosphere 71:765–772. doi: 10.1016/j.chemosphere.2007.10.013 CrossRefGoogle Scholar
  68. Weyens N, van der Lelie D, Taghavi S, Vangronsveld J (2009) Phytoremediation: plant-endophyte partnerships take the challenge. Curr Opin Biotechnol 20:248–254. doi: 10.1016/j.copbio.2009.02.012 CrossRefGoogle Scholar
  69. Wu X, Monchy S, Taghavi S, Zhu W, Ramos J, van der Lelie D (2011) Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida. FEMS Microbiol Rev 35:299–323. doi: 10.1111/j.1574-6976.2010.00249.x CrossRefGoogle Scholar
  70. Wuana RA, Okieimen FE (2011) Heavy metals in contaminated soils: a review of sources, chemistry, risks and best available strategies for remediation. ISRN Ecol 2011:1–20. doi: 10.5402/2011/402647 CrossRefGoogle Scholar
  71. Xu Y, Zhou Y, Ruan J, Xu S, Gu J, Huang S, Zheng L, Yuan B, Wen L (2015) Endogenous nitric oxide in Pseudomonas fluorescens ZY2 as mediator against the combined exposure to zinc and cefradine. Ecotoxicology 24:835–843. doi: 10.1007/s10646-015-1428-6 CrossRefGoogle Scholar
  72. Yamamoto S, Kasai H, Arnold DL, Jackson RW, Vivian A, Harayama S (2000) Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. Microbiology 146:2385–2394. doi: 10.1099/00221287-146-10-2385 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Biology Department and CESAMUniversity of AveiroAveiroPortugal
  2. 2.Biology Department, CESAM and iBiMEDUniversity of AveiroAveiroPortugal

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