Skip to main content

Advertisement

SpringerLink
Log in

Dynamics of endophytic bacteria in plant in vitro culture: quantification of three bacterial strains in Prunus avium in different plant organs and in vitro culture phases

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Endophytic bacteria occurring in plant in vitro cultures have often been described as contaminants, although these are generally present in all plant tissues, often with plant growth promoting effects. The effects of bacterial endophytes in different in vitro culture phases and in different plant organs of Prunus avium were studied. In a previous study we investigated the endophytic bacterial community of six registered silvaSELECT® genotypes and found differences in the bacterial community that correlated with propagation success. In this study, quantitative polymerase chain reaction protocols were developed to look at the dynamics of the most abundant endophytes, Mycobacterium spp., Rhodopseudomonas spp., and Microbacterium spp. These endophytes were quantified during propagation and rooting, and the bacterial content in three successive years was evaluated depicting the fluctuation over time. Leaves, stems, and shoots were found to contain bacteria although in different abundance. It was shown that after regeneration via adventitious shoots the bacteria were not eliminated, but showed slightly modified concentrations. The plant growth promoting traits of the two isolates Rhodopseudomonas palustris N-I-2 and Microbacterium testaceum D-I-1 were tested in an inoculation experiment, and showed a promotion in rooting of two difficult-to-propagate P. avium genotypes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

ITS:

16S to 23S internal transcribed spacer

NAA:

1-Naphthaleneacetic acid

qPCR:

Quantitative polymerase chain reaction

TDZ:

Thidiazuron

WPM:

Woody plant medium

References

  • 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 26:555–560. doi:10.1007/s11274-009-0191-3

    Article  Google Scholar 

  • 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 25:1757–1764. doi:10.1007/s11274-009-0073-8

    Article  Google Scholar 

  • Andreote F, Azevedo J, Araújo W (2009) Assessing the diversity of bacterial communities associated with plants. Braz J Microbiol 40:417–432. doi:10.1590/S1517-83822009000300001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Boine B, Naujoks G, Stauber T (2008) Investigations on influencing plant-associated bacteria in tissue cultures of black locust (Robinia pseudoacacia L.). Plant Cell Tissue Organ Cult 94:219–223. doi:10.1007/s11240-008-9395-8

    Article  CAS  Google Scholar 

  • Cardinale M, Grube M, Erlacher A, Quehenberger J, Berg G (2015) Bacterial networks and co-occurrence relationships in the lettuce root microbiota. Environ Microbiol 17:239–252. doi:10.1111/1462-2920.12686

    Article  CAS  PubMed  Google Scholar 

  • Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42:D633–D642. doi:10.1093/nar/gkt1244

    Article  CAS  PubMed  Google Scholar 

  • Compant S, Reiter B, Sessitsch A, Clément C, Barka EA, Nowak J (2005) Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Appl Environ Microbiol 71:1685–1693. doi:10.1128/AEM.71.4.1685

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dias ACF, Costa FEC, Andreote FD, Lacava PT, Teixeira MA, Assumpção LC, Araújo WL, Azevedo JL, Melo IS (2008) Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion. World J Microbiol Biotechnol 25:189–195. doi:10.1007/s11274-008-9878-0

    Article  Google Scholar 

  • Donnarumma F, Capuana M, Vettori C, Petrini G, Giannini R, Indorato C, Mastromei G (2011) Isolation and characterisation of bacterial colonies from seeds and in vitro cultures of Fraxinus spp. from Italian sites. Plant Biol 13:169–176. doi:10.1111/j.1438-8677.2010.00334.x

    Article  CAS  PubMed  Google Scholar 

  • Driver JA, Kuniyuki AH (1984) In vitro propagation of Paradox walnut root stock. Hort Sci 19:507–509

    Google Scholar 

  • Faleiro AC, Pereira TP, Espindula E, Cristiano F, Brod A, Brod FCA, Arisi ACM (2013) Real time PCR detection targeting nifA gene of plant growth promoting bacteria Azospirillum brasilense strain FP2 in maize roots. Symbiosis 61:125–133. doi:10.1007/s13199-013-0262-y

    Article  CAS  Google Scholar 

  • Ghyselinck J, Pfeiffer S, Heylen K, Sessitsch A, De Vos P (2013) The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies. PLoS One 8:e71360. doi:10.1371/journal.pone.0071360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • González-Rodríguez RM, Serrato R, Molina J, Aragón CE, Olalde V, Pulido LE, Dibut B, Lorenzo JC (2013) Biochemical and physiological changes produced by Azotobacter chroococcum (INIFAT5 strain) on pineapple in vitro-plantlets during acclimatization. Acta Physiol Plant 35:3483–3487. doi:10.1007/s11738-013-1373-z

    Article  Google Scholar 

  • Hardoim PR, Van Overbeek LS, Berg G, Pirttilä M, Compant S, Campisano A, Döring M (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320. doi:10.1128/MMBR.00050-14

    Article  PubMed  PubMed Central  Google Scholar 

  • Janßen A, Meier-Dinkel A, Steiner W, Degen B (2010) Forstgenetische Ressourcen der Vogel-Kirsche/Forest genetic resources of wild cherry. Forst und Holz 65:19–24

    Google Scholar 

  • Ji SH, Gururani MA, Chun S-C (2014) Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars. Microbiol Res 169:83–98. doi:10.1016/j.micres.2013.06.003

    Article  CAS  PubMed  Google Scholar 

  • Kobliha J (2002) Wild cherry (Prunus avium L.) breeding program aimed at the use of this tree in the Czech forestry. J For Sci 48:202–218

    Google Scholar 

  • Koh R-H, Song H-G (2007) Effects of application of Rhodopseudomonas sp. on seed germination and growth of tomato under axenic conditions. J Microbiol Biotechnol 17:1805–1810

    CAS  PubMed  Google Scholar 

  • Koskimäki JJ, Hankala E, Suorsa M, Nylund S, Pirttilä AM (2010) Mycobacteria are hidden endophytes in the shoots of rock plant [Pogonatherum paniceum (Lam.) Hack.] (Poaceae). Environ Microbiol Rep 2:619–624. doi:10.1111/j.1758-2229.2010.00197.x

    Article  PubMed  Google Scholar 

  • Lacava PT, Li WB, Arau WL, Azevedo JL, Hartung JS (2006) Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants. J Microbiol Methods 65:535–541. doi:10.1016/j.mimet.2005.09.015

    Article  CAS  PubMed  Google Scholar 

  • Laimer da Mâchado M, Heinrich M, Hanzer V, Arthofer W, Strommer S, Paltrinieri S, Martini M, Bertaccini A, Kummert J, Davies D (2001) Improved detection of viruses and phytoplasmas in fruit tree tissue cultures. Acta Hortic 550(463–470):2001. doi:10.17660/ActaHortic.550.70

    Google Scholar 

  • Laukkanen H, Soini H, Kontunen-Soppela S, Hohtola A, Viljanen M (2000) A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth of Scots pine seedlings. Tree Physiol 20:915–920. doi:10.1093/treephys/20.13.915

    Article  PubMed  Google Scholar 

  • Leifert C, Cassells A (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol Plant 37:133–138. doi:10.1079/IVP2000129

    Article  Google Scholar 

  • Lloyd G, McCown B (1980) Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. Comb Proc Int Plant Prop Soc 30:421–427

    Google Scholar 

  • Louden BC, Lynne AM, Haarmann D (2011) Use of blue agar CAS assay for siderophore detection. J Microbiol Biol Educ 12:51–53. doi:10.1128/jmbe.v12i1.249

    Article  PubMed  PubMed Central  Google Scholar 

  • Marino BG, Gaggìa F (2015) Antimicrobial activity of Melia azedarach fruit extracts for control of bacteria in inoculated in vitro shoots of “MRS 2/5” plum hybrid and calla lily and extract influence on the shoot cultures. Eur J Plant Pathol 141:505–521. doi:10.1007/s10658-014-0559-6

    Article  CAS  Google Scholar 

  • Marques JM, da Silva TF, Vollu RE, Blank AF, Ding G-C, Seldin L, Smalla K (2014) Plant age and genotype affect the bacterial community composition in the tuber rhizosphere of field-grown sweet potato plants. FEMS Microbiol Ecol 88:424–435. doi:10.1111/1574-6941.12313

    Article  CAS  PubMed  Google Scholar 

  • Matt A, Jehle JA (2005) In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.). Plant Cell Rep 24:468–476. doi:10.1007/s00299-005-0964-6

    Article  CAS  PubMed  Google Scholar 

  • Meier-Dinkel A (1986) In vitro Vermehrung ausgewählter Genotypen der Vogelkirsche (Prunus avium L.). Allg Forst-u J-Ztg 157:139–144

    Google Scholar 

  • Meier-Dinkel A, Steiner W, Artes O, Hosius B, Leinemann L (2007) Die silvaSELECT-Vogelkirschen- Klonmischung „Escherode I“. AFZ-Der Wald 5:246–247

    Google Scholar 

  • Middlebrook G, Cohn ML (1958) Bacteriology of tuberculosis: laboratory methods. Am J Public Health Nations Health 48:844–853

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moreno-Vázquez S, Larrañaga N, Uberhuaga EC, Braga EJB, Pérez-Ruíz C (2014) Bacterial contamination of in vitro plant cultures: confounding effects on somaclonal variation and detection of contamination in plant tissues. Plant Cell Tissue Organ Cult. doi:10.1007/s11240-014-0553-x

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Peralta KD, Araya T, Valenzuela S, Sossa K, Martínez M, Peña-Cortés H, Sanfuentes E (2012) Production of phytohormones, siderophores and population fluctuation of two root-promoting rhizobacteria in Eucalyptus globulus cuttings. World J Microbiol Biotechnol 28:2003–2014. doi:10.1007/s11274-012-1003-8

    Article  CAS  PubMed  Google Scholar 

  • 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 66:3073–3077. doi:10.1128/AEM.66.7.3073-3077.2000.Updated

    Article  PubMed  PubMed Central  Google Scholar 

  • Pirttilä AM, Joensuu P, Pospiech H, Jalonen J, Hohtola A, Pirttilä A, Joensuu P, Pospiech H, Jalonen J, Hohtola A (2004) Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology and mitigate browning of callus cultures. Physiol Plant 121:305–312. doi:10.1111/j.1399-3054.2004.00330.x

    Article  PubMed  Google Scholar 

  • Pirttilä AM, Pospiech H, Laukkanen H, Myllylä R, Hohtola A (2005) Seasonal variations in location and population structure of endophytes in buds of Scots pine. Tree Physiol 25:289–297

    Article  PubMed  Google Scholar 

  • Quadt-Hallmann A, Hallmann J, Kloepper JW (1997) Bacterial endophytes in cotton: location and interaction with other plant-associated bacteria. Can J Microbiol 43:254–259

    Article  CAS  Google Scholar 

  • Quambusch M, Pirttilä AM, Tejesvi MV, Winkelmann T, Bartsch M (2014) Endophytic bacteria in plant tissue culture: differences between easy- and difficult-to-propagate Prunus avium genotypes. Tree Physiol 34:524–533. doi:10.1093/treephys/tpu027

    Article  CAS  PubMed  Google Scholar 

  • Rahman A, Sitepu IR, Tang S-Y, Hashidoko Y (2010) Salkowski’s reagent test as a primary screening index for functionalities of rhizobacteria isolated from wild dipterocarp saplings growing naturally on medium-strongly acidic tropical peat soil. Biosci Biotechnol Biochem 74:2202–2208. doi:10.1271/bbb.100360

    Article  CAS  PubMed  Google Scholar 

  • R Development Core Team (2008) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

    Google Scholar 

  • Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant Microbe Interact 19:827–837. doi:10.1094/MPMI-19-0827

    Article  CAS  PubMed  Google Scholar 

  • Ruppel S, Rühlmann J, Merbach W (2006) Quantification and localization of bacteria in plant tissues using quantitative real-time PCR and online emission fingerprinting. Plant Soil 286:21–35. doi:10.1007/s11104-006-9023-5

    Article  CAS  Google Scholar 

  • Schmid T (2006) Prunus avium. In: Roloff A, Weisgerber H, Lang U, Stimm B (eds) Enzyklopädie der Holzgewächse. Wiley, Weinheim, pp 1–16

    Google Scholar 

  • Schulz BJE, Boyle CJC, Sieber TN (eds) (2006) What are endophytes? In: Microbial root endophytes. Springer, Berlin, pp 1–13

  • Shakya M, Gottel N, Castro H, Yang ZK, Gunter L, Labbe J, Muchero W, Bonito G, Vilgalys R, Tuskan G, Podar M, Schadt CW, Labbé J, Muchero W, Bonito G, Vilgalys R, Tuskan G, Podar M, Schadt CW (2013) A multifactor analysis of fungal and bacterial community structure in the root microbiome of mature Populus deltoides trees. PLoS One 8:e76382. doi:10.1371/journal.pone.0076382

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Taber RA, Thielen MA, Falkinham JO, Smith RH (1991) Mycobacterium scrofulaceum: a bacterial contaminant in plant tissue culture. Plant Sci 78:231–236. doi:10.1016/0168-9452(91)90203-K

    Article  Google Scholar 

  • Tang H, Ren Z, Reustle G, Krczal G, Reustle È, Krczal G (2002) Plant regeneration from leaves of sweet and sour cherry cultivars. Sci Hortic 93:235–244. doi:10.1016/S0304-4238(01)00328-4

    Article  CAS  Google Scholar 

  • Thomas P, Swarna GK, Roy PK, Patil P (2008) Identification of culturable and originally non-culturable endophytic bacteria isolated from shoot tip cultures of banana cv. Grand Naine. Plant Cell Tissue Organ Cult 93:55–63. doi:10.1007/s11240-008-9341-9

    Article  Google Scholar 

  • Trivedi P, Duan Y, Wang N (2010) Huanglongbing, a systemic disease, restructures the bacterial community associated with citrus roots. Appl Environ Microbiol 76:3427–3436. doi:10.1128/AEM.02901-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsavkelova EA, Cherdyntseva TA, Netrusov AI (2005) Auxin production by bacteria associated with orchid roots. Microbiology 74:46–53. doi:10.1007/s11021-005-0027-6

    Article  CAS  Google Scholar 

  • Ulrich K, Stauber T, Ewald D (2008) Paenibacillus—a predominant endophytic bacterium colonising tissue cultures of woody plants. Plant Cell Tissue Organ Cult 93:347–351. doi:10.1007/s11240-008-9367-z

    Article  Google Scholar 

  • Van den Houwe I, Guns J, Swennen R (1998) Bacterial contamination in Musa shoot tip cultures. Acta Hortic 490:485–492

    Article  Google Scholar 

  • Yang HY, Schmidt H (1992) Untersuchungen zur Adventivsproßregeneration in vitro bei Kirschen II. Adventivsproßbildung an in vitro-Blättern verschiedener Prunus avium-Idiotypen. Gartenbauwiss 57:7–10

    Google Scholar 

  • Zakhia F, Jeder H, Willems A, Gillis M, Dreyfus B, De Lajudie P (2006) Diverse bacteria associated with root nodules of spontaneous legumes in Tunisia and first report for nifH-like gene within the genera Microbacterium and Starkeya. Microb Ecol 51:375–393. doi:10.1007/s00248-006-9025-0

    Article  PubMed  Google Scholar 

  • Zaspel I, Ulrich A, Boine B, Stauber T (2008) Occurrence of culturable bacteria living in micropropagated black locust cultures (Robinia pseudoacacia L.). Eur J Hortic Sci 73:231–235

    Google Scholar 

Download references

Acknowledgments

The authors thank the Institut für Pflanzenkultur, Schnega, Germany, for providing the plant material and for motivating cooperative work, and our technicians Bärbel Ernst, Ewa Schneider and Friederike Schröder for their support. Financial support of the German Federal Ministry of Economics and Technology within the program PRO INNO Grant No. KF2508004AJ0 is gratefully acknowledged. M. Q. is a member of the graduate school WeGa Ph.D. funded by the German Federal Ministry of Education and Research and the Ministry for Science and Culture of Lower Saxony.

Author contributions

Conceived and designed the experiments: MQ, TW, MB. Performed the experiments: MQ, JB, KH. Analyzed the data: MQ, JB, KH, TW, MB. Wrote the paper: MQ, TW, MB.

Author information

Authors and Affiliations

  1. Woody Plant and Propagation Physiology Section, Institute of Horticultural Production Systems, Leibniz Universität Hannover, Herrenhaeuser Str. 2, 30419, Hannover, Germany

    Mona Quambusch, Jane Brümmer, Kristin Haller, Traud Winkelmann & Melanie Bartsch

Authors
  1. Mona Quambusch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jane Brümmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kristin Haller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Traud Winkelmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Melanie Bartsch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Melanie Bartsch.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1Supplementary material 2 (PDF 153 kb)

Supplementary material 3

Determination of the detection limit for the three primer sets to measure a Mycobacterium spp. b Microbacterium spp. and c Rhodopseudomonas spp. in P. avium in vitro material. Dark blue = Standard curve; light blue = Standard curve with 10 ng cherry DNA added to the reaction; green = plant DNA as positive control (for Mycobacterium spp. one with high, one with low bacterial DNA content); grey = control cherry DNA (without the measured bacterium); black = no template control. Red arrow marks the defined detection limit, grey arrow marks the control cherry DNA (PDF 172 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Quambusch, M., Brümmer, J., Haller, K. et al. Dynamics of endophytic bacteria in plant in vitro culture: quantification of three bacterial strains in Prunus avium in different plant organs and in vitro culture phases. Plant Cell Tiss Organ Cult 126, 305–317 (2016). https://doi.org/10.1007/s11240-016-0999-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-016-0999-0

Keywords

Search

Navigation