Skip to main content
SpringerLink
Log in

Purification of Substances from Achyrocline satureioides with Inhibitory Activity Against Paenibacillus larvae, the Causal Agent of American Foulbrood in Honeybees’ Larvae

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Achyrocline satureioides extracts were tested in vitro against the growth of Paenibacillus larvae. Four different extracts were obtained by liquid-liquid extraction from an aqueous-ethyl alcohol macerate of the aerial parts of the plant. The biological activity was tested by the broth microdilution technique. Hexane extract showed the highest activity (minimum inhibitory concentration = 0.060 ± 0.037 mg/mL). Transmission electron microscopy experiments showed that the main effect exerted by the hexane extract on the cell was at the cellular membrane level. The hexane extract was analyzed by thin-layer chromatography, and the activity of its components was tested by bioautography. Four growth inhibition zones were observed in the bioautographic experiments (using hexane-acetone (7:3) as mobile phase), with that at Rf = 0.57 showing the largest zone of inhibition. High-performance liquid chromatographic experiments, using ultraviolet and electrospray ionization coupled to tandem mass spectrometric detection, showed the presence of one compound with a m/z ratio of 442, which may be related to phloroglucinols α-pyrone compounds recently discovered. The high antibacterial activity of the hexane extract and of the isolated compound determined in this work may be useful for the development of future new alternatives for the treatment of American foulbrood.

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

Similar content being viewed by others

References

  1. Genersh, E. (2010). American Foulbrood in honeybees and its causative agent, Paenibacillus larvae. Journal of Invertebrate Pathology, 103, S10–S19.

    Article  Google Scholar 

  2. Alippi, A. M. (1996). Caracterización de aislamientos de Paenibacillus larvae mediante tipo bioquímico y resistencia a oxitetraciclina. Revista Argentina de Microbiologia, 28, 197–203.

    CAS  Google Scholar 

  3. Alippi, A. M., López, A. C., Reynaldi, F. J., Grasso, D. H., & Aguilar, O. M. (2007). Evidence for plasmid-mediated tetracycline resistance in Paenibacillus larvae, the causal agent of American Foulbrood (AFB) disease in honeybees. Veterinary Microbiology, 125, 290–303.

    Article  CAS  Google Scholar 

  4. Evans, J. D. (2003). Diverse origins of tetracycline resistance in the honey bee bacterial pathogen, Paenibacillus larvae. Journal of Invertebrate Pathology, 83, 46–50.

    Article  CAS  Google Scholar 

  5. Miyagi, T., Peng, C. Y. S., Chuang, R. Y., Mussen, E. C., Spivak, M. S., & Doi, R. H. (2000). Verification of oxytetracycline-resistant American Foulbrood pathogen Paenibacillus larvae in the United States. Journal of Invertebrate Pathology, 75, 95–96.

    Article  CAS  Google Scholar 

  6. Bastos, E. M. A. F., Simone, M., Jorge, D. M., Soares, A. E. E., & Spivak, M. (2008). In vitro study of the antimicrobial activity of Brazilian propolis against Paenibacillus larvae. Journal of Invertebrate Pathology, 97, 273–281.

    Article  CAS  Google Scholar 

  7. Antúnez, K., Harriet, J., Gende, L., Maggi, M., Eguaras, M., & Zunino, P. (2008). Efficacy of natural propolis extract in the control of American Foulbrood. Veterinary Microbiology, 131, 324–331.

    Article  Google Scholar 

  8. Alippi, A. M., Ringuelet, J. A., Cerimele, E. L., Re, M. S., & Henning, C. P. (1996). Antimicrobial activity of some essential oils against Paenibacillus larvae, the causal agent of American Foulbrood disease. Journal of Herbs, Spices and Medicinal Plants, 4, 9–16.

    Article  Google Scholar 

  9. Floris, I., Carta, C., & Moretti, M. D. L. (1996). Activités in vitro de plusieurs huiles essentielles sur Bacillus larvae White et essai au rucher. Apidologie, 27, 111–119.

    Article  Google Scholar 

  10. Fuselli, S. R., Garcia de la Rosa, S., Gende, L. B., Eguaras, M. J., & Fritz, R. (2006). Antimicrobial activity of some Argentinean wild plant essential oils against Paenibacillus larvae larvae, causal agent of American Foulbrood (AFB). Journal of Apicultural Research, 45, 2–7.

    Article  CAS  Google Scholar 

  11. Fuselli, S. R., Garcia de la Rosa, S. B., Eguaras, M. J., & Fritz, R. (2008). Chemical composition and antimicrobial activity of Citrus essences on honeybee bacterial pathogen Paenibacillus larvae, the causal agent of American Foulbrood. World Journal of Microbiology and Biotechnology, 24, 2067–2072.

    Article  CAS  Google Scholar 

  12. Gende, L. B., Floris, I., Fritz, R., & Eguaras, M. J. (2008). Antimicrobial activity of cinnamon (Cinnamomum zeylanicum) essential oil and its main components against Paenibacillus larvae from Argentine. Bulletin of Insectology, 61, 1–4.

    Google Scholar 

  13. Gende, L. B., Fernández, N., Buffa, F., Ruiu, L., Satta, A., Fritz, R., Eguaras, M. J., & Floris, I. (2010). Susceptibility of Paenibacillus larvae isolates to a tetracycline hydrochloride and cinnamon (Cinnamomum zeylanicum) essential oil mixture. Bulletin of Insectology, 63, 247–250.

    Google Scholar 

  14. Vianna Santos, R. C., dos Santos Alves, C. F., Schneider, T., Soares Lopes, L. Q., Aurich, C., Luehring Giongo, J., Brandelli, A., & de Almeida, V. R. (2012). Antimicrobial activity of Amazonian oils against Paenibacillus species. Journal of Invertebrate Pathology, 109, 265–268.

    Article  Google Scholar 

  15. Sabaté, D. C., Carrillo, L., & Audisio, M. C. (2009). Inhibition of Paenibacillus larvae and Ascosphaera apis by Bacillus subtilis isolated from honeybee gut and honey samples. Research in Microbiology, 160, 163–169.

    Article  Google Scholar 

  16. Audisio, M. C., Torres, M. J., Sabaté, D. C., Ibarguren, C., & Apella, M. C. (2011). Properties of different lactic acid bacteria isolated from Apis mellifera L. bee-gut. Microbiological Research, 166, 1–13.

    Article  Google Scholar 

  17. Evans, J. D., & Armstrong, T. N. (2005). Inhibition of the American foulbrood bacterium, Paenibacillus larvae, by bacteria isolated from honey bees. Journal of Apicultural Research, 44, 168–171.

    CAS  Google Scholar 

  18. Yoshiyama, M., & Kimura, K. (2009). Bacteria in the gut of Japanese honeybee, Apis cerana japonica, and their antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood. Journal of Invertebrate Pathology, 102, 91–96.

    Article  Google Scholar 

  19. Alippi, A. M., & Reynaldi, F. J. (2006). Inhibition of the growth of Paenibacillus larvae, the causal agent of American Foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources. Journal of Invertebrate Pathology, 91, 141–146.

    Article  Google Scholar 

  20. Nascimento, G. G. F., Locatelli, J., Freitas, P. C., & Silva, G. L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. Brazilian Journal of Microbiology, 31, 247–256.

    Google Scholar 

  21. Albo, G. N., Henning, C., Ringuelet, J. A., Reynaldi, F. J., De Giusti, M. R., & Alippi, A. M. (2003). Evaluation of some essential oils for the control and prevention of American Foulbrood disease in honey bees. Apidologie, 34, 417–437.

    Article  CAS  Google Scholar 

  22. Gende, L. B., Maggi, M. D., Fritz, R., Eguaras, M. J., Bailac, P., & Ponzi, M. (2009). Antimicrobial activity of Pimpinella anisum and Foeniculum vulgare essential oils against Paenibacillus larvae. Journal of Essential Oil Research, 21, 91–93.

    Article  CAS  Google Scholar 

  23. González, M. J., & Marioli, J. M. (2010). Antibacterial activity of water extracts and essential oils of various aromatic plants against Paenibacillus larvae, the causative agent of American Foulbrood. Journal of Invertebrate Pathology, 104, 209–213.

    Article  Google Scholar 

  24. Retta, D., Dellacassa, E., Villamil, J., Suárez, S., & Bandoni, L. (2012). Marcela, a promising medicinal and aromatic plant from Latin America: A review. Industrial Crops and Products, 38, 27–38.

    Article  Google Scholar 

  25. Alippi, A. M. (1990). Técnicas de laboratorio para el aislamiento e identificación de Bacillus larvae White, agente causal de la Loque Americana. Buenos Aires: Comisión de Investigaciones Científicas, Serie difusión. Año 2, N° 6.

    Google Scholar 

  26. Mann, C. M., & Markham, J. L. (1998). A new method for determining the minimum inhibitory concentration of essential oils. Journal of Applied Microbiology, 84, 538–544.

    Article  CAS  Google Scholar 

  27. Choma, I. M., & Grzelak, E. M. (2011). Bioautography detection in thin-layer chromatography. Journal of Chromatography A, 1218, 2684–2691.

    Article  CAS  Google Scholar 

  28. Gende, L. B., Principal, J., Maggi, M. D., Palacios, S. M., Fritz, R., & Eguaras, M. J. (2008). Extracto de Melia azedarach y aceites esenciales de Cinnamomum zeylanicum, Mentha piperita y Lavandula officinalis como control de Paenibacillus larvae. Zootecnia Tropical, 26, 151–156.

    Google Scholar 

  29. Reynaldi, F. J., Rule, R., Arauz, S., & Alippi, A. M. (2010). Sensibilidad in vitro de Paenibacillus larvae frente a los antibióticos oxitetraciclina, tilosina, tilmicosina y lincomicina. Analecta Veterinaria, 30, 25–29.

    Google Scholar 

  30. Sikkema, J., de Bont, J. A. M., & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiological Reviews, 59, 201–222.

    CAS  Google Scholar 

  31. Bakkali, F., Averbeck, S., Averbeck, D., & Idaomar, M. (2008). Biological effects of essential oils—a review. Food and Chemical Toxicology, 46, 446–475.

    Article  CAS  Google Scholar 

  32. Casero, C. (2012). Metabolitos secundarios de Achyrocline satureioides. Quimiomodulación de la actividad antimicrobiana (dissertation). Argentina: Universidad Nacional de Río Cuarto.

    Google Scholar 

  33. Rocha, L., Marsston, A., Potterat, O., Kaplan, A. C., Stoeckli-Evans, H., & Hostettmann, K. (1995). Antibacterial phloroglucinols and flavonoids from Hypericum brasiliense. Phytochemistry, 40, 1447–1452.

    Article  CAS  Google Scholar 

  34. Joray, M. B., Rollán, M. C., Ruiz, G. M., Palacios, S. M., & Carpinella, M. C. (2011). Antibacterial activity of extracts from plants of central Argentina—isolation of an active principle from Achyrocline satureioides. Planta Medica, 77, 95–100.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We gratefully appreciate financial support from SeCyT-Universidad Nacional de Córdoba, SeCyT-Universidad Nacional de Río Cuarto, Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), and Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET). MJG thanks CONICET for her doctoral fellowship. MCA and JMM are members of Carrera del Investigador Científico de CONICET (CIC-CONICET).

Author information

Authors and Affiliations

  1. Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Río Cuarto, Argentina

    María J. González & Viviana G. Beoletto

  2. IMBIV-CONICET and Departamento de Farmacia, Universidad Nacional de Córdoba, Córdoba, Argentina

    Alicia M. Agnese

  3. Instituto de Investigaciones para la Industria Química (INIQUI-CONICET), Salta, Argentina

    Marcela C. Audisio

  4. Departamento de Química, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, ruta 36 km 601, X5804BYA, Río Cuarto, Argentina

    Juan M. Marioli

Authors
  1. María J. González
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Viviana G. Beoletto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alicia M. Agnese
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcela C. Audisio
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan M. Marioli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juan M. Marioli.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

González, M.J., Beoletto, V.G., Agnese, A.M. et al. Purification of Substances from Achyrocline satureioides with Inhibitory Activity Against Paenibacillus larvae, the Causal Agent of American Foulbrood in Honeybees’ Larvae. Appl Biochem Biotechnol 175, 3349–3359 (2015). https://doi.org/10.1007/s12010-015-1506-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1506-5

Keywords

Search

Navigation