Molecular identification and control of endophytic contamination during in vitro plantlet development of Fagonia indica

Abstract

Microbial contamination is the major cause of economic losses in commercial and scientific plant tissue culture laboratories. For successful micropropagation, it is important to control contamination during in vitro cultures. The present study was designed to isolate, identify and eradicate endophytic contaminants from in vitro cultures of medicinally important plant Fagonia indica. A total of eight distinct bacterial isolates from in vitro grown plantlets of F. indica were selected based on analysis of colony morphology. The endophytic bacterial contaminants identified at the species level through 16S rRNA sequence analysis were Enterobacter xiangfangensis, Bacillus vallismortis, Bacillus tequilensis, Terribacillus halophilus, Pantoea dispersa, Serratia marcescens subsp. Sakuensis, Staphylococcus epidermidis and Bacillus atrophaeus. It was observed that almost 60% of seedlings were contaminated with Bacillus sp. and out of those, Bacillus tequelensis contributed to most infections (70% out of the Bacillus infections). The other most frequently occurring bacteria were Bacillus vallismortis, Terribacillus halophilus and Serratia marcescens subsp. sakuensis. Furthermore, the addition of antimicrobials to the media either completely inhibited or drastically decreased the growth of endophytic bacteria as compared to the control in which 92% of the plantlets were contaminated with these endophytes. Nine different antibiotics (rifampicin, teicoplanin, gentamicin, vancomycin, ciprofloxacin, tobramycin, tetracycline, doxycycline and ampicillin) were tested for their activity against the identified endophytes. Antibiotics such as ciprofloxacin and tobramycin showed a good response and inhibited the growth of all the bacterial isolates at low doses compared to the other antibiotics. Tobramycin was the most effective as it inhibited the growth of five of the bacterial isolates at a dosage as low as 4 mg/L. In case of tetracycline (16 mg/L) and doxycycline (64 mg/L), the contamination frequency in plantlets was 25.6 and 45%, respectively. It is, therefore, important to search for more endophytes, causing adverse effects during in vitro cultures and should devise a feasible anti-microbial strategy for controlling such contamination.

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

Fig. 1
Fig. 2

References

  1. Abbasi BH, Khan MA, Mahmood T, Ahmad M, Chaudhary MF, Khan MA (2010) Shoot regeneration and free-radical scavenging activity in Silybum marianum L. Plant Cell Tiss Org Cult 101:371–376. https://doi.org/10.1007/s11240-010-9692-x

    Article  CAS  Google Scholar 

  2. Afzal I, Shinwari ZK, Iqrar I (2015) Selective isolation and characterization of agriculturally beneficial endophytic bacteria from wild hemp using canola. Pak J Bot 47:1999–2008

    CAS  Google Scholar 

  3. Afzal I, Iqrar I, Shinwari ZK, Yasmin A (2016) Plant growth-promoting potential of endophytic bacteria isolated from roots of wild Dodonaea viscosa L. Plant Growth Regul. https://doi.org/10.1007/s10725-016-0216-5

    Article  Google Scholar 

  4. Bagban I, Roy S, Chaudhary A, Das S, Gohil K, Bhandari K (2012) Hepatoprotective activity of the methanolic extract of Fagonia indica Burm in carbon tetra chloride induced hepatotoxicity in albino rats. Asian Pac J Trop Biomed 2:S1457–S1460

    Article  Google Scholar 

  5. Bibi F (2017) Diversity of antagonistic bacteria isolated from medicinal plant Peganum harmala L. Saudi J Biol Sci 24:1288–1293. https://doi.org/10.1016/j.sjbs.2015.09.021

    Article  PubMed  CAS  Google Scholar 

  6. Brunner I, Echegaray A, Rubluo A (1995) Isolation and characterization of bacterial contaminants from Dieffenbachia amoena Bull, Anthurium andreanum Linden and Spathiphyllum sp. Schoot cultured in vitro. Sci Hortic 62:103–111. https://doi.org/10.1016/0304-4238(94)00743-y

    Article  Google Scholar 

  7. Cao M, Moore CM, Helmann JD (2005) Bacillus subtilis paraquat resistance is directed by sigmaM, an extracytoplasmic function sigma factor, and is conferred by YqjL and BcrC. J Bacteriol 187:2948–2956. https://doi.org/10.1128/JB.187.9.2948-2956.2005

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Chen W, Yeh D (2007) Elimination of in vitro contamination, shoot multiplication, and ex vitro rooting of Aglaonema. HortScience 42:629–632

    CAS  Google Scholar 

  9. CLSI (2007) Performance standards for antimicrobial susceptibility testing. Seventeenth information supplement. Clinical and Laboratory Standards Institute, West Valley Road, PA, USA

    Google Scholar 

  10. 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. https://doi.org/10.1007/s11274-008-9878-0

    Article  CAS  Google Scholar 

  11. Dienstag J, Neu HC (1972) In vitro studies of tobramycin. an aminoglycoside antibiotic. Antimicrob Agents Chemother 1:41–45

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. 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. https://doi.org/10.1111/j.1438-8677.2010.00334.x

    Article  PubMed  CAS  Google Scholar 

  13. Falkiner F (1987) Strategy for the selection of antibiotics for use against common bacterial pathogens and endophytes of plants. Bacterial and bacteria-like contaminants of plant tissue cultures. Acta Hort 225:53–56

    Google Scholar 

  14. Fang J-Y, Hsu Y-R (2012) Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures. Plant Cell Tiss Org Cult 110:53–62. https://doi.org/10.1007/s11240-012-0129-6

    Article  CAS  Google Scholar 

  15. Holland MA, Polacco JC (1994) PPFMs and other covert contaminants: is there more to plant physiology than just plant? Annu Rev Plant Physiol Plant Mol Biol 45:197–209. https://doi.org/10.1146/annurev.pp.45.060194.001213

    Article  CAS  Google Scholar 

  16. Hooker JD (1897) The flora of British India, vol v. 7. L. Reeve, The New York Public Library, Ashford, Kent

    Google Scholar 

  17. Khan MA, Abbasi BH, Ali H, Ali M, Adil M, Hussain I (2015) Temporal variations in metabolite profiles at different growth phases during somatic embryogenesis of Silybum marianum L. Plant Cell Tiss Org Cult 120:127–139

    Article  CAS  Google Scholar 

  18. Khan T, Abbasi BH, Khan MA, Azeem M (2017) Production of biomass and useful compounds through elicitation in adventitious root cultures of Fagonia indica Ind Crops. Prod 108:451–457. https://doi.org/10.1016/j.indcrop.2017.07.019

    CAS  Article  Google Scholar 

  19. Kneifel W, Leonhardt W (1992) Testing of different antibiotics against gram-positive and gram-negative bacteria isolated from plant tissue culture. Plant Cell Tiss Org Cult 29:139–144. https://doi.org/10.1007/bf00033619

    Article  CAS  Google Scholar 

  20. Kotra LP, Haddad J, Mobashery S (2000) Aminoglycosides: perspectives on mechanisms of action and resistance and strategies to counter resistance. Antimicrob Agents Chemother 44:3249–3256. https://doi.org/10.1128/aac.44.12.3249-3256.2000

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Kritzinger EM, van Vuuren RJ, Woodward B, Rong IH, Spreeth MH, Slabbert MM (1997) Elimination of external and internal contaminants in rhizomes of Zantedeschia Aethiopica with commercial fungicides and antibiotics. In: Cassells AC (ed) Pathogen and microbial contamination management in micropropagation. Developments in plant pathology, vol 12. Springer, Netherlands, pp 161–167. https://doi.org/10.1007/978-94-015-8951-2_19

    Google Scholar 

  22. Kumar P, Dubey RC, Maheshwari DK (2012) Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens. Microbiol Res 167:493–499. https://doi.org/10.1016/j.micres.2012.05.002

    Article  PubMed  CAS  Google Scholar 

  23. Kunisaki J (1980) In vitro propagation of. Anthurium andreanum Lind. HortScience 15:508–509

    Google Scholar 

  24. Lam M, Wolff K, Griffiths H, Carmichael A (2014) Correction: an aqueous extract of Fagonia cretica induces DNA damage, cell cycle arrest and apoptosis in breast cancer cells via FOXO3a and p53 expression. PLoS One. https://doi.org/10.1371/journal.pone.0102655

    Article  PubMed  PubMed Central  Google Scholar 

  25. Lata H, Li XC, Silva B, Moraes RM, Halda-Alija L (2006) Identification of IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA sequencing. Plant Cell Tiss Org Cult 85:353–359. https://doi.org/10.1007/s11240-006-9087-1

    Article  CAS  Google Scholar 

  26. Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol Plant 37:133–138. https://doi.org/10.1007/s11627-001-0025-y

    Article  Google Scholar 

  27. Leifert C, Waites WM (1992) Bacterial growth in plant tissue culture media. J Appl Bacteriol 72:460–466. https://doi.org/10.1111/j.1365-2672.1992.tb01859.x

    Article  Google Scholar 

  28. Leifert C, Woodward S (1997) Laboratory contamination management; the requirement for microbiological quality assurance. In: Cassells AC (ed) Pathogen and microbial contamination management in micropropagation. Developments in Plant Pathology. Springer, Dordrecht, pp 237–244. https://doi.org/10.1007/978-94-015-8951-2_30

    Google Scholar 

  29. Leifert C, Ritchie JY, Waites WM (1991) Contaminants of plant-tissue and cell cultures. World J Microbiol Biotechnol 7:452–469. https://doi.org/10.1007/BF00303371

    Article  PubMed  CAS  Google Scholar 

  30. Luna C, Collavino M, Mroginski L, Sansberro P (2008) Identification and control of bacterial contaminants from Ilex dumosa nodal segments culture in a temporal immersion bioreactor system using 16S rDNA analysis. Plant Cell Tiss Org Cult 95:13–19. https://doi.org/10.1007/s11240-008-9408-7

    Article  CAS  Google Scholar 

  31. Luna C, Acevedo R, Collavino M, González A, Mroginski L, Sansberro P (2013) Endophytic bacteria from Ilex paraguariensis shoot cultures: localization, characterization, and response to isothiazolone biocides. In Vitro Cell Dev Biol Plant 49:326–332. https://doi.org/10.1007/s11627-013-9500-5

    Article  CAS  Google Scholar 

  32. McManus PS, Stockwell VO, Sundin GW, Jones AL (2002) Antibiotic use in plant agriculture. Annu Rev Phytopathol 40:443–465. https://doi.org/10.1146/annurev.phyto.40.120301.093927

    Article  PubMed  CAS  Google Scholar 

  33. Mehta R, Champney WS (2002) 30S ribosomal subunit assembly is a target for inhibition by aminoglycosides in Escherichia coli. Antimicrob Agents Chemother 46:1546–1549. https://doi.org/10.1128/aac.46.5.1546-1549.2002

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Mingeot-Leclercq MP, Glupczynski Y, Tulkens PM (1999) Aminoglycosides: activity and resistance. Antimicrob Agents Chemother 43:727–737

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  35. Misra P, Gupta N, Toppo DD, Pandey V, Mishra MK, Tuli R (2009) Establishment of long-term proliferating shoot cultures of elite Jatropha curcas L. by controlling endophytic bacterial contamination. Plant Cell Tiss Org Cult 100:189–197. https://doi.org/10.1007/s11240-009-9636-5

    Article  Google Scholar 

  36. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

    Article  CAS  Google Scholar 

  37. Nagorska K, Bikowski M, Obuchowski M (2007) Multicellular behaviour and production of a wide variety of toxic substances support usage of Bacillus subtilis as a powerful biocontrol agent. Acta Biochim Pol 54:495–508

    PubMed  CAS  Google Scholar 

  38. Pareek A, Godavarthi A, Issarani R, Nagori BP (2013) Antioxidant and hepatoprotective activity of Fagonia schweinfurthii (Hadidi) Hadidi extract in carbon tetrachloride induced hepatotoxicity in HepG2 cell line and rats. J Ethnopharmacol 150:973–981. https://doi.org/10.1016/j.jep.2013.09.048

    Article  PubMed  Google Scholar 

  39. Rasamiravaka T, Labtani Q, Duez P, El Jaziri M (2015) The formation of biofilms by Pseudomonas aeruginosa: a review of the natural and synthetic compounds interfering with control mechanisms. BioMed Res Int 2015:759348. https://doi.org/10.1155/2015/759348

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Rashid S, Charles TC, Glick BR (2012) Isolation and characterization of new plant growth-promoting bacterial endophytes. Appl Soil Ecol 61:217–224. https://doi.org/10.1016/j.apsoil.2011.09.011

    Article  Google Scholar 

  41. Reed BM, Buckley PM, DeWilde TN (1995) Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cell Dev Biol Plant 31:53–57. https://doi.org/10.1007/bf02632228

    Article  Google Scholar 

  42. Saeed S, Ali H, Khan T, Kayani W, Khan MA (2017) Impacts of methyl jasmonate and phenyl acetic acid on biomass accumulation and antioxidant potential in adventitious roots of Ajuga bracteosa Wall ex Benth., a high valued endangered medicinal plant. Physiol Mol Biol Plants 23:229–237. https://doi.org/10.1007/s12298-016-0406-7

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Saleem S, Jafri L, ul Haq I, Chang LC, Calderwood D, Green BD, Mirza B (2014) Plants Fagonia cretica L. and Hedera nepalensis K. Koch contain natural compounds with potent dipeptidyl peptidase-4 (DPP-4) inhibitory activity. J Ethnopharmacol 156:26–32. https://doi.org/10.1016/j.jep.2014.08.017

    Article  PubMed  CAS  Google Scholar 

  44. Shehata AM, Wannarat W, Skirvin RM, Norton MA (2010) The dual role of carbenicillin in shoot regeneration and somatic embryogenesis of horseradish (Armoracia rusticana) in vitro. Plant Cell Tiss Org Cult 102:397–402. https://doi.org/10.1007/s11240-010-9732-6

    Article  CAS  Google Scholar 

  45. Shen H, Li Z, Han D, Yang F, Huang Q, Ran L (2010) Detection of indigenous endophytic bacteria in Eucalyptus urophylla in vitro conditions. Front Agric China 4:37–41. https://doi.org/10.1007/s11703-009-0090-2

    Article  Google Scholar 

  46. Waheed A, Barker J, Barton SJ, Owen CP, Ahmed S, Carew MA (2012) A novel steroidal saponin glycoside from Fagonia indica induces cell-selective apoptosis or necrosis in cancer cells. Eur J Pharm Sci 47:464–473. https://doi.org/10.1016/j.ejps.2012.07.004

    Article  PubMed  CAS  Google Scholar 

  47. Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163–175. https://doi.org/10.1038/nprot.2007.521

    Article  PubMed  CAS  Google Scholar 

  48. Young LS, Hewitt WL (1973) Activity of five aminoglycoside antibiotics in vitro against gram-negative bacilli and Staphylococcus aureus. Antimicrob Agents Chemother 4:617–625. https://doi.org/10.1128/aac.4.6.617

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

Tariq Khan acknowledges the indigenous Ph.D. fellowship program of Higher Education Commission, Pakistan. Dr. Bilal Haider Abbasi acknowledges financial support from Pakistan Academy of Sciences, Pakistan.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Tariq Khan or Bilal Haider Abbasi.

Ethics declarations

Conflict of interest

All the authors (TK, BA, II, MA, and ZK) declare that they have no conflict of interest.

Additional information

Communicated by M. Lambardi.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Khan, T., Abbasi, B.H., Iqrar, I. et al. Molecular identification and control of endophytic contamination during in vitro plantlet development of Fagonia indica. Acta Physiol Plant 40, 150 (2018). https://doi.org/10.1007/s11738-018-2727-3

Download citation

Keywords

  • Endophytes
  • Contaminants
  • Antibiotics
  • Fagonia
  • Germination