Advertisement

Use of antibiotics to control endophytic bacterial growth migration onto culture medium in Eucalyptus cloeziana F.Muell.: a micropropagation approach

  • Gabriela Ferraz LeoneEmail author
  • Pedro Avelino Maia Andrade
  • Carolina Vieira de Almeida
  • Cristina Vieira de Almeida
  • Fernando Dini Andreote
  • Marcílio de Almeida
Plant Tissue Culture
  • 52 Downloads

Abstract

The natural endophytic bacterial growth migration onto a culture medium is commonly associated with unnecessary microplant discards. In micropropagation procedures, some bacteria can exude from the internal tissues of plants to colonize the culture medium and compete with the plants for nutrients, which may lead to a reduction in plant development. To find an efficient antibiotic protocol to control this bacterial growth migration onto the culture medium without affecting plant development, Eucalyptus cloeziana F.Muell. microstumps were subjected to four antibiotic treatments for 30 d. They were treated with gentamicin, ciprofloxacin, rifampicin, or Timentin®, in addition to the control treatment (antibiotic free). The effects of the antibiotics were monitored weekly, and the endophytic bacterial community structures were evaluated in two periods of plant development (15 d and 30 d).The denaturing gradient gel electrophoresis (DGGE) technique was used to compare the control and post-antibiotic treatment plant microbiological composition, to determine if the antibiotic treatment played a specific role on the endophytic bacterial community structure. The gentamicin treatment was composed of a distinct community from the control treatment. Nonetheless, the plants treated with ciprofloxacin and rifampicin manifested similar endophytic community structures compared to the control. In contrast, plants treated with Timentin® showed a specific bacterial community composition and a higher plant dry mass, number of shoots, and nutritional content. These results suggested that Timentin® treatment could be applied for 30 days to control endophytic bacterial growth migration onto the culture medium, without affecting the homeostatic balance between the bacteria and plants.

Keywords

In vitro culture Microplants Endophytes Antibiotic therapy Eucalyptus 

Notes

Funding Information

The São Paulo Research Foundation (FAPESP; Process No. 2011/15861-6) supported this research.

References

  1. Abreu-Tarazi MF, Navarrete AA, Andreote FD, Almeida CV, Tsai SM, Almeida M (2010) Endophytic bacteria in long-term in vitro cultivated “axenic” pineapple microplants revealed by PCR-DGGE. World J Microbiol Biotechnol 26:555–560CrossRefGoogle Scholar
  2. Almeida CV, Andreote FD, Yara R, Tanaka FAO, Azevedo JL, Almeida M (2009) Bacteriosomes in axenic plants: endophytes as stable endosymbionts. World J Microbiol Biotechnol 25:1757–1764CrossRefGoogle Scholar
  3. Almeida CV, Yara R, Almeida M (2005) Endophytic fungi in shoot tip of the pejibaye cultivated in vivo and in vitro. Pesq Agrop Brasileira 40:467–470CrossRefGoogle Scholar
  4. Andreote FD, Gumiere T, Durrer A (2014) Exploring interactions of plant microbiomes. Sci Agric 71:528–539CrossRefGoogle Scholar
  5. Azevedo JL, Maccheroni Junior W, Pereira JO, Araújo WL (2000) Endophytic microorganisms: a review on insect control and recent advances on tropical plants. Electron J Biotechnol 3:40–65CrossRefGoogle Scholar
  6. Baccarin FJB, Brondani GE, Almeida LV, Vieira IG, Oliveira LS, Almeida M (2015) Vegetative rescue and cloning of Eucalyptus benthamii selected adult trees. New For 46:465–483CrossRefGoogle Scholar
  7. Baldotto LEB, Baldotto MA, Olivares FL, Viana AP, Bressan-Smith R (2010) Selection of growth-promoting bacteria for pineapple ‘Vitória’ during acclimatization. Rev Bras Ciênc Solo 34:349–360CrossRefGoogle Scholar
  8. Berg G (2009) Plant–microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture. App Microbiol Biotechnol 84:11–18CrossRefGoogle Scholar
  9. Berg G, Grube M, Schloter M, Smalla K (2014) Unraveling the plant microbiome: looking back and future perspectives. Front Microbiol 5:1–7Google Scholar
  10. Bouffaud ML, Poirier MA, Muller D, Moënne-Loccoz Y (2014) Root microbiome relates to plant host evolution in maize and other Poaceae. Environ Microbiol 16:2804–2814CrossRefGoogle Scholar
  11. Bray JR, Curtis JT (1957) An ordination of the upland forest communities of southern Wisconsin. Ecol Monogr 27:325–349CrossRefGoogle Scholar
  12. Brody JR, Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. BioTechniques 36:214–216CrossRefGoogle Scholar
  13. Brondani GE, Baccarin FJB, Gonçalves AN, Almeida M (2014) Nutritional content in Eucalyptus benthamii mini-stump leaves. Acta Sci Agron 36:465–474CrossRefGoogle Scholar
  14. Brondani GE, Ondas HWW, Baccarin FJB, Gonçalves AN, Almeida M (2012) Micropropagation of Eucalyptus benthamii to form a clonal micro-garden. In Vitro Cell Dev Biol Plant 48:35–48CrossRefGoogle Scholar
  15. Chanprame S, Todd JJ, Widholm JM (1996) Prevention of pink-pigmented methylotrophic bacteria (Methylobacterium mesophilicum) contamination of plant tissue cultures. Plant Cell Rep 16:222–225CrossRefGoogle Scholar
  16. Chelius MK, Triplett EW (2001) The diversity of archaea and bacteria in association with the roots of Zea mays L. Microb Ecol 41:252–263CrossRefGoogle Scholar
  17. Cheng ZM, Schnurr JA, Kapaun JA (1998) Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Rep 17:646–649CrossRefGoogle Scholar
  18. Costa MGC, Nogueira FTS, Figueira ML, Otoni WC, Brommonschenkel SH, Cecon PR (2000) Influence of the antibiotic timentin on plant regeneration of tomato (Lycopersicon esculentum Mill.) cultivars. Plant Cell Rep 19:327–332CrossRefGoogle Scholar
  19. Cunha ACMCM, Paiva HN, Leite HG, Barros NF, Leite FP (2009) Relações entre variáveis climáticas com produção e enraizamento de miniestacas de eucalipto. R Árvore 33:195–203CrossRefGoogle Scholar
  20. Delaporte KL, Conran JG, Sedgley M (2001) Interspecific hybridization within Eucalyptus (myrtaceae): subgenus symphyomyrtus, sections Bisectae and Adnataria. Int J Plant Sci 162(6):1317–1326CrossRefGoogle Scholar
  21. Dutra LF, Wendling I, Brondani GE (2009) A micropropagação de eucalipto. Pesqui Florest Bras 58:49–59Google Scholar
  22. Esposito-Polesi NP, Abreu-Tarazi MF, Almeida CV, Tsai SM, Almeida M (2017) Investigation of endophytic bacterial community in supposedly axenic cultures of pineapple and orchids with evidence on abundant intracellular bacteria. Curr Microbiol 74:103–113CrossRefGoogle Scholar
  23. Esposito-Polesi NP, Andrade PAM, Almeida CV, Andreote FD, Almeida M (2015) Endophytic bacterial communities associated with two explant sources of Eucalyptus benthamii Maiden & Cambage. World J Microbiol Biotechnol 31:1737–1746CrossRefGoogle Scholar
  24. Fang JY, Hsu YR (2012) Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures. Plant Cell Tissue Organ Cult 110:53–62CrossRefGoogle Scholar
  25. Fellner M (1995) Influence of the antibiotic ciprofloxacin on culture of Allium longicuspis callus-derived protoplasts. Ann Bot 76:219–223CrossRefGoogle Scholar
  26. Gai CS, Dini-Andreote F, Andreote FD, Lopes JRS, Araújo WL, Miller TA, Azevedo JL, Lacava PT (2011) Endophytic bacteria associated to sharpshooters (Hemiptera: Cicadellidae), insect vectors of Xylella fastidiosa subsp. pauca. J Plant Pathol Microbiol 2:2–8Google Scholar
  27. Hammer O, Harper DAT, Ryan PD (2001) PAST: paleontological statistical software package for education and data analysis. Palaeontol Electron 4:1–9Google Scholar
  28. Hardoim PR, Van Overbeek LS, Van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471CrossRefGoogle Scholar
  29. Heuer H, Krsek M, Baker P, Smalla K, Wellington EM (1997) Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-eletrophoretic separation in denaturing gradients. Appl Environ Microbiol 63:3233–3241Google Scholar
  30. Izumi H, Moore ERB, Killham K, Alexander IJ, Anderson IC (2007) Characterization of endobacterial communities in ectomycorrhizas by DNA- and RNA-based molecular methods. Soil Biol Biochem 39:891–899CrossRefGoogle Scholar
  31. Jucoski GO, Cambraia J, Ribeiro C, Oliveira JA (2016) Excess iron on growth and mineral composition in Eugenia uniflora L. Ciênc Agron 47:720–728Google Scholar
  32. 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 6:1244–1251CrossRefGoogle Scholar
  33. Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol Plant 37:133–138CrossRefGoogle Scholar
  34. Leone GF, Almeida CV, Abreu-Tarazi MF, Batagin-Piotto KD, Almeida M (2016) Antibiotic therapy in pineapple (Ananas comosus) microplants. Ciênc Rural 46:89–94CrossRefGoogle Scholar
  35. Lloyd G, McCown B (1981) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Comb Proc Int Plant Prop Soc 30:421–427Google Scholar
  36. MacArthur RH, Wilson EO (1967) The theory of island biogeography. Princeton University Press, Princeton 203pGoogle Scholar
  37. Malavolta E, Vitti GC, Oliveira AS (1997) Methodology for analysis of elements in plant material of the book, assessment of the nutritional status of plants: principles and applications, 2nd edn.ed., see. and current. POTAFOS, PiracicabaGoogle Scholar
  38. Mediavilla A, Flórez J, García-Lobo JM (1997) Pharmacology of infectious diseases: general principles, selection and associations of antibiotics. In: Human pharmacology, 5th edn. Santander Elsevier, Barcelona 1479pGoogle Scholar
  39. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Planta 15(3):473–497Google Scholar
  40. Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel eletrophoresis analysis of polymerase chain reaction-amplified genes encoding for 16S rRNA. App Environ Microbiol 59:695–700Google Scholar
  41. Nauerby B, Billing K, Wyndaele R (1997) Influence of the antibiotic timentin on plant regeneration compared to carbenicillin and cefotaxime in concentrations suitable for elimination of Agrobacterium tumefaciens. Plant Sci 123:169–177CrossRefGoogle Scholar
  42. Nehra NS, Becwar MR, Rottmann WH, Pearson L, Chowdhury K, Chang S, Wilde HD, Kodrzycki RJ, Zhang C, Gause KC, Parks DW, Hinchee MA (2005) Forest biotechnology: innovative methods, emerging opportunities. In Vitro Cell Dev Biol Plant 41:701–717CrossRefGoogle Scholar
  43. Nübel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI, Ludwig W, Backhaus H (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643CrossRefGoogle Scholar
  44. Oberschelp GPJ, Gonçalves AN (2016) Assessing the effects of basal media on the in vitro propagation and nutritional status of Eucalyptus dunnii Maiden. In Vitro Cell Dev Biol Plant 52:28–37CrossRefGoogle Scholar
  45. Oliveira LS, Brondani GE, Batagin-Piotto KD, Calsavara R, Gonçalves AN, Almeida M (2015) Micropropagation of Eucalyptus cloeziana mature trees. Aust For 78:219–231CrossRefGoogle Scholar
  46. Oliveira LS, Xavier A, Lopes AP, Takahashi EK, Otoni WC (2016) Multiplicação e alongamento in vitro de clones híbridos de Eucalyptus globulus. Ci Fl 26:235–247CrossRefGoogle Scholar
  47. Oliveira MLP, Costa MGC, Silva CV, Otoni WC (2010) Growth regulators, culture media and antibiotics in the in vitro shoot regeneration from mature tissue of citrus cultivars. Pesq Agrop Brasileira 45:654–660CrossRefGoogle Scholar
  48. Orlikowska T, Nowak K, Reed B (2017) Bacteria in the plant tissue culture environment. Plant Cell Tissue Organ Cult 128:487–508CrossRefGoogle Scholar
  49. Pamphile JA, Azevedo JL (2002) Molecular characterization of endophytic strains of Fusarium verticillioides (Fusarium moniliforme) from maize (Zea mays L.). World J Microbiol Biotechnol 18:391–396CrossRefGoogle Scholar
  50. Panicker B, Thomas P, Janakiran T, Venugopalan R, Narayanappa SB (2007) Influence of cytokinin levels on in vitro propagation of shy suckering chrysanthemum “Arka Swarna” and activation of endophytic bacteria. In Vitro Cell Dev Biol Plant 43:614–622CrossRefGoogle Scholar
  51. Pereira JES, Mattos MLT, Fortes GLR (2003) Identification and antibiotic control of the endophytic bacteria contaminants in micropropagated potato explants. Pesq Agrop Brasileira 38:827–834CrossRefGoogle Scholar
  52. Pirttilä AM, Podolich O, Koskimäki JJ, Hohtola E, Hohtola A (2008) Role of origin and endophyte infection in browning of bud-derived tissue cultures of Scots pine (Pinus sylvestris L.). Plant Cell Tissue Organ Cult 95:47–55CrossRefGoogle Scholar
  53. Quecine MC, Araújo WL, Rossetto PB, Ferreira A, Tsui S, Lavaca PT, Mondin M, Azevedo JL, Pizzirani-Kleiner AA (2012) Sugarcane growth promotion by the endophytic bacterium Pantoea agglomerans 33.1. App Environ Microbiol 78:7511–7518CrossRefGoogle Scholar
  54. R Development Core Team (2005) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna www.R-project.orgAccessed 15 Dec 2018
  55. Ramage CM, Williams RR (2002) Mineral nutrition and plant morphogenesis. In Vitro Cell Dev Biol Plant 38:115–124CrossRefGoogle Scholar
  56. Rana F (2013) Rifampicin—an overview. Int J Res Pharm Chem 3:83–877Google Scholar
  57. Reinhold-Hurek B, Hurek T (1998) Interactions of gramineous plants with Azoarcus spp. and other diazotrophs: identification, localization, and perspectives to study their function. Crit Rev Plant Sci 17:29–54CrossRefGoogle Scholar
  58. Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant-Microbe Interact 19:827–837CrossRefGoogle Scholar
  59. Sarruge JR, Haag HP (1974) Chemical analysis in plants. ESALQ/USP, Piracicaba 56pGoogle Scholar
  60. Silva JAT, Nhut DT, Tanaka M, Fukai S (2003) The effect of antibiotics on the in vitro growth response of chrysanthemum and tobacco stem transverse thin cell layers (tTCLs). Sci Hortic 97:397–410CrossRefGoogle Scholar
  61. Silveira RLVA (2004) Evaluation of the nutritional status of eucalypts: visual and foliar diagnosis and their interpretation. In: JLM G, Benedetti V (eds) Forest nutrition and fertilization. IPEF Piracicaba, Brazil, pp 85–111Google Scholar
  62. Stape JL, Gonçalves JLM, Gonçalves AN (2001) Relationships between nursery practices and field performance for Eucalyptus plantations in Brazil. New For 22:19–41CrossRefGoogle Scholar
  63. Street HE (2014) Plant metabolism, 3rd edn. Electronic Resource: online access, 321p. https://library.plantandfood.co.nz/cgi-bin/koha/opac-search.pl?q=au:%22Cockburn,%20W.,%22. Acessed 09 July 2017
  64. Taiz L, Zeiger E (2013) Plant physiology, 4th edn. Sinauer Associates Inc., Publishers Sunderland, Massachusetts 954pGoogle Scholar
  65. Tambarussi EV, Rogalski M, Nogueira FTS, Brondani GE, De Martin VF, Carrer H (2015) Influence of antibiotics on indirect organogenesis of teak. Ann For Res 58:177–183CrossRefGoogle Scholar
  66. ter Braak CJF (1990) CANOCO: a Fortran program for canonical community ordination; update notes. Agricultural Mathematics Group, WageningenGoogle Scholar
  67. Thomas P, Prakash GS (2004) Sanitizing long-term micropropagated grapes from covert and endophytic bacteria and preliminary field testing of plants after 8 years in vitro. In Vitro Cell Dev Biol Plant 40:603–607CrossRefGoogle Scholar
  68. Thomas P, Swarna GK, Patil P, Rawal RD (2008a) Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures. Plant Cell Tissue Organ Cult 93:39–54CrossRefGoogle Scholar
  69. Thomas P, Swarna GK, Roy PK, Patil P (2008b) Identification of culturable and originally nonculturable endophytic bacteria isolated from shoot tip cultures of banana cv. Grand Naine. Plant Cell Tissue Organ Cult 93:55–63CrossRefGoogle Scholar
  70. Turner TR, Ramakrishnan K, Walshaw J, Heavens D, Alston M, Swarbreck D, Osbourn A, Grant A, Poole PS (2013) Comparative metatranscriptomics reveals kingdom level changes in the rhizosphere microbiome of plants. ISME J 7:2248–2258CrossRefGoogle Scholar
  71. Vacheron J, Desbrosses G, Bouffaud ML, Touraine B, Moënne-Loccoz Y, Muller D, Legendre L, Wisniewski-Dyé F, Prigent-Combaret C (2013) Plant growth-promoting rhizobacteria and root system functioning. Front Plant Sci 4:356CrossRefGoogle Scholar
  72. Vandenkoornhuyse P, Quaiser A, Duhamel M, Le Van A, Dufresne A (2015) The importance of the microbiome of the plant holobiont. New Phytol 206(4):1196–1206CrossRefGoogle Scholar
  73. Yasuhara T, Nokihara AK (2001) High-throughput analysis of total nitrogen content that replaces the classic Kjeldahl method. J Agric Food Chem 49:4581–4583CrossRefGoogle Scholar
  74. Zanuncio JC, Mezzomo JA, Guedes RNC, Oliveira AC (1998) Influence of strips of native vegetation on Lepidoptera associated with Eucalyptus cloeziana in Brazil. For Ecol Manag 108:85–90CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Gabriela Ferraz Leone
    • 1
    Email author
  • Pedro Avelino Maia Andrade
    • 2
  • Carolina Vieira de Almeida
    • 3
  • Cristina Vieira de Almeida
    • 4
  • Fernando Dini Andreote
    • 2
  • Marcílio de Almeida
    • 1
  1. 1.Department of Biological Science, “Luiz de Queiroz” College of Agriculture (ESALQ)University of Sao Paulo (USP)PiracicabaBrazil
  2. 2.Department of Soil Science, “Luiz de Queiroz” College of Agriculture (ESALQ)University of Sao Paulo (USP)PiracicabaBrazil
  3. 3.Dipartimento di Medicina Sperimentale e ClinicaUniversità Degli Studio FirenzeFlorenceItaly
  4. 4.InVitroPalm (Consulting, Study and Biological Development Ltda.)PiracicabaBrazil

Personalised recommendations