Plant and Soil

, Volume 334, Issue 1–2, pp 461–474

Diversity of endophytic bacteria from the cuprophytes Haumaniastrum katangense and Crepidorhopalon tenuis

  • Alfred Cubaka Kabagale
  • Bertrand Cornu
  • Françoise van Vliet
  • Claire-Lise Meyer
  • Max Mergeay
  • Jean-Baptiste Lumbu Simbi
  • Louis Droogmans
  • Corinne Vander Wauven
  • Nathalie Verbruggen
Regular Article

Abstract

Haumaniastrum katangense and Crepidorhopalon tenuis are two cuprophytes characteristic of the Katangan Copper Belt flora. We have studied the endophytic bacteria of H. katangense and C. tenuis as a first step to evaluate their potential contribution to plant adaptation to copper excess. Although their number varied considerably from sample to sample, culturable bacteria were found in roots and shoots of most plants. More than 800 isolates were screened for each plant species. Identification of isolates based on the sequence of the 16S rRNA gene, allocated them to 31 taxonomic units, belonging to 17 genera, mainly Proteobacteria. A great proportion of the bacteria were cupro-resistant and often resistant to other metals, especially zinc and cobalt, as well as nickel for the Methylobacterium isolates. Direct PCR amplification of the polymorphic bacterial internal transcribed spacer (ITS) from the plants’ organs DNA revealed a more diverse endophytic community, with more Gram+, among which a Rubrobacteridae that was never found associated with plants before. This work represents the first study of endophytes in Katangan cuprophytes.

Keywords

Copper tolerance Haumaniastrum katangense Crepidorhopalon tenuis Endophytic bacteria 

Supplementary material

11104_2010_396_MOESM1_ESM.doc (36 kb)
Table S1Number of plant specimens containing endophytic isolates from the same taxonomic group as the mentioned isolate. +, presence of isolate; −, absence of isolate. n = 7 (H. katangense) and 6 (C. tenuis). Results from Pearson’s chi square exact tests are indicated for each taxon. Isolates were considered for the statistical analysis when they were detected more than once (DOC 34 kb).
11104_2010_396_MOESM2_ESM.doc (52 kb)
Table S2Distribution between plant species and among organs of the ITS amplicons obtained from more than one plant-extracted DNA sample. +, number of DNA samples from which the sequence was obtained; −, number of samples from which it was not obtained. n = 8 for H. katangense; n = 6 for C. tenuis leaves and stems and n = 4 for C. tenuis roots. No significant differences were found between species. Significant differences among organs are in bold and indicated as follows: **, P < 0.001; *, P < 0.05. Ampl. for amplicon (DOC 47 kb).

References

  1. Altschul SF, Madden TL, Schaffer 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 25:3389–3402CrossRefPubMedGoogle Scholar
  2. Baker A, Brooks R, Pease A, Malaisse F (1983) Studies on copper and cobalt tolerance in three closely related taxa within the genus Silene L. (Caryophyllaceae) from Zaïre. Plant Soil 73:377–385CrossRefGoogle Scholar
  3. Barac T, Taghavi T, Borremans B, Provoost A, Oeyen L, Colpaert JV, Vangronsveld J, van der Lelie D (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble volatile organic pollutants. Nat Biotechnol 22:583–588CrossRefPubMedGoogle Scholar
  4. Barzanti R, Ozino F, Bazzicalupo M, Gabbrielli R, Galardi F, Gonnelli C, Mengoni A (2007) Isolation and characterization of endophytic bacteria from the nickel hyperaccumulator plant Alyssum bertolonii. Microb Ecol 53:306–316CrossRefPubMedGoogle Scholar
  5. Brim H, Heyndrickx M, De Vos P, Wilmotte A, Springael D, Schlegel HG, Mergeay M (1999) Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs. Syst Appl Microbiol 22:258–268PubMedGoogle Scholar
  6. Brooks RR (1978) Copper and cobalt uptake by Haumaniastrum species. Plant Soil 48:541–544CrossRefGoogle Scholar
  7. Burkhead JL, Gogolin-Reynolds KA, Abdel-Ghany SA, Cohu C, Pilon M (2009) Copper homeostasis. New Phytol. doi:10.1111/j.1469-8137.2009.02846 PubMedGoogle Scholar
  8. Bussman I, Philipp B, Schink B (2001) Factors influencing the cultivability of lake water bacteria. J Microbiol Meth 47:41–50CrossRefGoogle Scholar
  9. Cardinale M, Brusetti L, Quatrini P, Borin S, Puglia AM, Rizzi A, Zanardini C, Sorlini C, Corselli C, Daffonchio D (2004) Comparison of different primer sets for the automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. Appl Environ Microbiol 70:6147–6156CrossRefPubMedGoogle Scholar
  10. Chipeng F, Hermans C, Colinet G, Faucon MP, Ngongo M, Meert P, Verbruggen N (2010) Copper tolerance in the cuprophyte Haumaniastrum katangense (S. Moore) P. A. Duvign & Plancke. Plant Soil 328:235–244CrossRefGoogle Scholar
  11. Corpe WA, Rheem S (1989) Ecology of the methylotrophic bacteria on living leaf surfaces. FEMS Microbiol Ecol 62:243–249CrossRefGoogle Scholar
  12. Diels L, Mergeay M (1990) DNA probe-mediated detection of resistant bacteria from soils highly polluted by heavy metals. Appl Environ Microbiol 56:1485–1491PubMedGoogle Scholar
  13. Doty S (2008) Enhancing phytoremediation through the use of transgenics and endophytes. New Phytol 179:318–333CrossRefPubMedGoogle Scholar
  14. Duvigneaud P (1958) The vegetation of Katanga and its metalliferous soils. Bull Soc R Bot Belg 90:127–286Google Scholar
  15. Edwards U, Rogall T, Blocker H, Emde M, Bottger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853CrossRefPubMedGoogle Scholar
  16. Faucon M-P, Shutcha M, Meerts P (2007) Revisiting copper and cobalt concentrations in supposed hyperaccumulators from SC Africa: influence of washing and metal concentrations in soil. Plant Soil 301:29–36CrossRefGoogle Scholar
  17. Faucon M-P, Colinet G, Mahy G, Ngongo Luhembwe M, Verbruggen N, Meerts P (2008) Soil influence on Cu and Co uptake and plant size in the cuprophytes Crepidorhopalon perennis and C. tenuis (Scrophulariaceae) in SC Africa. Plant Soil 317:201–212CrossRefGoogle Scholar
  18. Ferreira AC, Nobre MF, Moore E, Rainey FA, Battista JR, da Costa M (1999) Characterization and radiation resistance of new isolates of Rubrobacter radiotolerans and Rubrobacter xylanophilus. Extremophiles 3:235–238CrossRefPubMedGoogle Scholar
  19. Fischer E (1989) Contributions of the flora of Central Africa III: new species of Lindernia Allioni and Crepidorhopalon E. Fisher (Scrophulariaceae) from Zaïre, Burundi and Tanzania. Bull Jard Bot Nat Belg 60:409–413CrossRefGoogle Scholar
  20. Gonzales N, Romero J, Espejo RT (2003) Comprehensive detection of bacterial populations by PCR amplification of the 16S–23S rRNA spacer region. J Microbiol Meth 55:91–97CrossRefGoogle Scholar
  21. Gürtler V, Stanisich VA (1996) New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiology 142:3–16CrossRefPubMedGoogle Scholar
  22. Hardoim P, van Overbeek L, van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471CrossRefPubMedGoogle Scholar
  23. Holmes A, Bowyer J, Holley M, O’Donoghue M, Montgomery M, Gillings MR (2000) Diverse, yet-to-be-cultured members of the Rubrobacter subdivision of the Actinobacteria are widespread in Australian arid soils. FEMS Microbiol Ecol 33:111–120CrossRefPubMedGoogle Scholar
  24. Idris R, Trifonova R, Puschenreiter M, Wenzel WW, Sessitsch A (2004) Bacterial communities associated with flowering plants of the Ni-hyperaccumulator Thlaspi goesingense. Appl Environ Microbiol 70:2667–2677CrossRefPubMedGoogle Scholar
  25. Idris R, Kuffner M, Bodrossy L, Puschenreiter M, Monchy S, Wenzel WW, Sessitsch A (2006) Characterization of Ni-tolerant methylobacteria associated with the hyperaccumulating plant Thlaspi goesingense and description of Methylobacterium goesingense sp. nov. Syst Appl Microbiol 29:634–644CrossRefPubMedGoogle Scholar
  26. Ikeda S, Fuji S-I, Sato T, Ytow N, Ezura H, Minamisawa K, Fujimura T (2006) Community analysis of seed-associated microbes in forage crops using culture-independent methods. Microbes Environ 21:112–121CrossRefGoogle Scholar
  27. Kuffner M, De Maria S, Puschenreiter M, Fallmann K, Wieshammer G, Gorfer M, Strauss J, Rivelli A, Sessitsch A (2010) Culturable bacteria from Zn- and Cd-accumulating Salix caprea with differential effects on plant growth and heavy metal availability. J Appl Microbiol. doi:10.1111/j.1365-2672.2010.04670.x PubMedGoogle Scholar
  28. Kutschera U (2007) Plant-associated methylobacteria as co-evolved phytosymbionts. Plant Signal Behav 2:74–78PubMedGoogle Scholar
  29. Leteinturier B, Baker AJ, Malaisse F (1999) Early stages of natural revegetation of metalliferous mine workings in South Central Africa: a preliminary survey. Biotechnol Agron Soc Environ 3:28–41Google Scholar
  30. Lodewyckx C, Taghavi S, Mergeay M, Vangronsveld J, Clijsters H, van der Lelie D (2001) The effect of recombinant heavy metal-resistant endophytic bacteria on heavy metal uptake by their host plant. Int J Phytoremediat 3:173–187CrossRefGoogle Scholar
  31. Lodewyckx C, Mergeay M, Vangronsfeld J, Clijsters H, van der Lelie D (2002) Isolation, characterization, and identification of bacteria associated with the zinc hyperaccumulator Thlaspi caerulescens subsp. calaminaria. Int J Phytoremediat 4:101–105CrossRefGoogle Scholar
  32. Macnair M (2003) The hyperaccumulation of metals by plants. Adv Bot Res 40:63–105CrossRefGoogle Scholar
  33. Malaisse F, Brooks RR (1982) Colonisation of modified metalliferous environments in Zaire by the copper flower Haumaniastrum katangense. Plant Soil 64:289–293CrossRefGoogle Scholar
  34. Malaisse F, Brooks RR, Baker AJ (1994) Diversity of vegetation communities in relation to soil heavy metal content at the Shinkolobwe copper/cobalt/uranium mineralization, upper Shaba, Zaire. Belg J Bot 127:3–16Google Scholar
  35. Marschner H (1995) Functions of mineral nutrients: micronutrients. In: Marschner H (ed) Mineral nutrition of higher plants. Academic Press, London, pp 313–404Google Scholar
  36. Mastretta C, Taghavi S, van der Lelie D, Mengoni A, Galardi F, Gonnelli C, Barac T, Boulet J, Weyens N, Vangronsveld J (2009) Endophytic bacteria from seeds of Nicotiana tabacum can reduce cadmium phytotoxicity. Int J Phytoremediat 11:251–267CrossRefGoogle Scholar
  37. McInroy JA, Kloepper JW (1995) Survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil 173:337–342CrossRefGoogle Scholar
  38. Meharg A (2005) Mechanisms of plant resistance to metal and metalloid ions and potential for biotechnology applications. Plant Soil 274:163–174CrossRefGoogle Scholar
  39. Mengoni A, Pini F, Huang L-N, Shu W-S, Bazzicalupo M (2009) plant-by-plant variations of bacterial communities associated with leaves of the nickel hyperaccumulator Alyssum bertolonii Desv. Microb Ecol 58:660–667CrossRefPubMedGoogle Scholar
  40. Mergeay M, Nies D, Schlegel HG, Gerits J, Charles P, Van Gijsegem F (1985) Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacteriol 162:328–334PubMedGoogle Scholar
  41. Monchy S, Benotmane MA, Wattiez R, van Aelst S, Auquier V, Borremans B, Mergeay M, Taghavi S, van der Lelie D, Vallaeys T (2006) Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34. Microbiology 152:1765–1776CrossRefPubMedGoogle Scholar
  42. Pirttilä AM, Laukkanen H, Pospiech H, Myllylä R, Hohtola A (2000) Detection of intracellular bacteria in the buds of Scotch pine (Pinus sylvestris L.) by in situ hybridization. Appl Environ Microbiol 66:3073–3077CrossRefPubMedGoogle Scholar
  43. Reeves RD (2003) Tropical hyperaccumulators of metals and their potential for phytoextraction. Plant Soil 249:57–65CrossRefGoogle Scholar
  44. Reeves RD (2006) Hyperaccumulation of trace elements by plants. In: Morel JL, Echevarria G, Goncharova N (eds) Phytoremediation of metal contaminated soils. NATO science series: IV: earth and environmental sciences, vol 68. Springer, New York, pp 25–52Google Scholar
  45. Reeves RD, Baker AJ (2000) Metal-accumulating plants. In: Raskin I, Ensley BD (eds) Phytoremediation of toxic metals: using plants to clean up the environment. Wiley, New York, pp 193–229Google Scholar
  46. Rosenblueth M, Martinez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant-Microb Interact 19:827–837CrossRefGoogle Scholar
  47. Ryan RP, Germain K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent developments and applications. FEMS Microbiol Lett 278:1–9CrossRefPubMedGoogle Scholar
  48. Saito A, Ikeda S, Ezura H, Minamisawa K (2007) Microbial community analysis of the phytosphere using culture-independent methodologies. Microbes Environ 22:93–105CrossRefGoogle Scholar
  49. Sheng X-F, Xia J-J, Jiang C-Y, He L-Y, Qian M (2008) characterization of heavy metal-resistant endophytic bacteria from rape (Brassica napus) roots and their potential in promoting the growth and lead accumulation of rape. Environ Pollut 156:1164–1170CrossRefPubMedGoogle Scholar
  50. Sun L-N, Zhang Y-F, He L-Y, Chen Z-J, Wang Q-Y, Qian M, Sheng X-F (2010) Genetic diversity and characterization of heavy metal-resistant endophytic bacteria from two copper-tolerant plant species on copper mine wasteland. Bioresour Technol 101:501–509CrossRefPubMedGoogle Scholar
  51. Surette M, Sturz AV, Lada RR, Nowak J (2003) Bacterial endophytes in processing carrots (Daucus carota L. var. sativus): their localization, population density, biodiversity and their effects on plant growth. Plant Soil 253:381–390CrossRefGoogle Scholar
  52. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMedGoogle Scholar
  53. Trotsenko YA, Ivanova EG, Doronina NV (2001) Aerobic methylotrophic bacteria as phyto-symbionts. Microbiology 70:623–632CrossRefGoogle Scholar
  54. Ulrich K, Ulrich A, Ewald D (2008) Diversity of endophytic bacterial communities in poplar grown under field conditions. FEMS Microbiol Ecol 63:169–180CrossRefPubMedGoogle Scholar
  55. Velázquez E, Rojas M, Lorite MJ, Rivas R, Zurdo-Piñeiro JL, Heydrich M, Bedmar E (2008) Genetic diversity of endophytic bacteria which could be found in the apoplastic sap of the medullary parenchym of the stem of healthy sugarcane plants. J Basic Microbiol 48:118–124CrossRefPubMedGoogle Scholar
  56. Verbruggen N, Hermans C, Schat H (2009) Molecular mechanisms of metal hyperaccumulation in plants. New Phytol 181:759–776CrossRefGoogle Scholar
  57. von Wintzingerode F, Göbel U, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21:213–229CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Alfred Cubaka Kabagale
    • 1
    • 5
    • 6
  • Bertrand Cornu
    • 2
  • Françoise van Vliet
    • 2
  • Claire-Lise Meyer
    • 1
  • Max Mergeay
    • 3
    • 4
  • Jean-Baptiste Lumbu Simbi
    • 5
  • Louis Droogmans
    • 3
  • Corinne Vander Wauven
    • 2
  • Nathalie Verbruggen
    • 1
  1. 1.Laboratoire de Physiologie et de Génétique Moléculaire des PlantesUniversité Libre de BruxellesBrusselsBelgium
  2. 2.Institut de Recherches Microbiologiques JMWBrusselsBelgium
  3. 3.Laboratoire de MicrobiologieUniversité Libre de BruxellesBrusselsBelgium
  4. 4.Molecular and Cellular Biology, Unit for MicrobiologyBelgian Center for Nuclear Energy (SCK*CEN)MolBelgium
  5. 5.Laboratoire de Chimie OrganiqueUniversité de LubumbashiKatangaDRC
  6. 6.Laboratoire de Physiologie Végétale et de Microbiologie AppliquéeUniversité Officielle de BukavuSud-KivuDRC

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