, Volume 44, Issue 1, pp 19–34 | Cite as

Analysis of the genetic diversity and phylogenetic relationships of Biscogniauxia mediterranea isolates associated with cork oak

  • Joana HenriquesEmail author
  • Filomena Nóbrega
  • Edmundo Sousa
  • Arlindo Lima


Biscogniauxia mediterranea is one of the most frequent fungal pathogens involved in cork oak decline in the Mediterranean Basin, causing charcoal canker. In Portugal, this disease is widespread on adult declining trees but nowadays it increasingly affects young trees and exhibits atypical symptoms, leading to the hypothesis that some change in the fungus may have occurred. In order to evaluate the genetic diversity and phylogenetic relationship of B. mediterranea associated with cork oak, 102 isolates were obtained from young and adult trees of Quercus suber and other hosts species with different disease expression, from several Mediterranean countries. The collection of isolates was analyzed by individual and multigene phylogenies using Maximum-Likelihood approach based on nucleotide sequences of the internal transcribed spacers of ribosomal DNA, translation elongation factor 1-α and β-tubulin genes, and by microsatellite-primed PCR profiles. Sequence analyses separated the Mediterranean isolates from those from other regions, while MSP-PCR analysis revealed relevant but unstructured diversity among the Mediterranean isolates under study, making this a monophyletic but diverse population. Considering the adaptive capacity of the fungus in the Mediterranean-climate ecosystems and the present climatic change scenario, all conditions are gathered to favor aggravation of the disease in cork oak stands.


Charcoal canker intraspecific variability Mediterranean ecosystem Quercus suber 



Authors are grateful to everyone who contributed to the sample collection and to Professor Manuel Mota for this manuscript revision. This research was partially supported by Fundação para a Ciência e a Tecnologia, Portugal (grant number SFRH/BD/46787/2008).

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Brown, J. K. M., & Hovmaller, M. S. (2002). Aerial Dispersal of Pathogens on the Global and Continental Scales and Its Impact on Plant Disease. Science, 297, 537–541.CrossRefPubMedGoogle Scholar
  2. Burdon, J. J., & Silk, J. (1997). Sources and Patterns of Diversity in Plant-Pathogenic Fungi. Phytopathology, 87(7), 664–669.CrossRefPubMedGoogle Scholar
  3. Câmara, M. S. (1930). Contributions ad Mycoflorum Lusitaniae. Centuriae VIII et IX. Anais Instituto Superior de Agronomia, 3, 84.Google Scholar
  4. Carbone, I., & Kohn, L. M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91(3), 553–556.CrossRefGoogle Scholar
  5. Cohen, S. D. (2005). A protocol for direct sequencing of multiple gene specific PCR products from Discula umbrinella, a fungal endophyte, utilizing bufferless precast electrophoresis. Journal of Microbiological Methods, 61(1), 131–135.CrossRefPubMedGoogle Scholar
  6. Collado, J., Platas, G., & Peláez, F. (2001). Identification of an endophytic Nodulisporium sp. from Quercus ilex in central Spain as the anamorph of Biscogniauxia mediterranea by rDNA sequence analysis and effect of different ecological factors on distribution of the fungus. Mycologia, 93(5), 875–886.CrossRefGoogle Scholar
  7. Cowling, R. M., Rundel, P. W., Lamont, B. B., Arroyo, M. K., & Arianoutsou, M. (1996). Plant diversity in mediterranean climate regions. Trends in Ecology & Evolution, 11(9), 362–366.CrossRefGoogle Scholar
  8. Diogo, E. L. F., Santos, J. M., & Philips, A. J. L. (2010). Phylogeny, morphology and pathogenicity of Diaporthe and Phomopsis species on almond in Portugal. Fungal Diversity, 44(1), 107–115.CrossRefGoogle Scholar
  9. Erwin, D. H. (2009). Climate as a Driver of Evolutionary Change. Current Biology, 19(14), R575–R583.CrossRefPubMedGoogle Scholar
  10. Glass, N. L., & Donaldson, G. C. (1995). Development of Primer Sets Designed for Use with the PCR To Amplify Conserved Genes from Filamentous Ascomycetes. Applied and Environmental Microbiology, 61(4), 1323–1330.PubMedCentralPubMedGoogle Scholar
  11. Henriques, J., Inácio, M. L., Lima, A., & Sousa, E. (2012). New outbreaks of charcoal canker on young cork oak trees in Portugal. IOBC/wprs Bulletin, 76, 85–88.Google Scholar
  12. Henriques, J., Barrento, M. J., Bonifácio, L., Gomes, A. A., Lima, A., & Sousa, E. (2014a). Factors affecting the dispersion of Biscogniauxia mediterranea in Portuguese cork oak stands. Silva Lusitana, 22(1), 83–97.Google Scholar
  13. Henriques, J., Nóbrega, F., Sousa, E., & Lima, A. (2014b). Diversity of Biscogniauxia mediterranea within single stromata on cork oak. Journal of Mycology, article ID 324349. doi: 10.1155/2014/324349.
  14. Hsieh, H., Ju, Y., & Rogers, J. D. (2005). Molecular phylogeny of Hypoxylon and closely related genera. Mycologia, 97(4), 844–865.CrossRefPubMedGoogle Scholar
  15. Inácio, M. L., Henriques, J., Guerra-Guimarães, L., Gil-Azinheira, H., Lima, A., & Sousa, E. (2011). Platypus cylindrus Fab. (Coleoptera: Platypodidae) transports Biscogniauxia mediterranea, agent of cork oak charcoal canker. Boletín de sanidad vegetal. Plagas, 37(2), 181–186.Google Scholar
  16. Jiménez, J. J., Sánchez, M. E., & Trapero, A. (2005a). El chancro carbonoso de Quercus I: Distribución y caracterización del agente casual. Boletín de sanidad vegetal. Plagas, 31(4), 549–562.Google Scholar
  17. Jiménez, J. J., Sánchez, M. E., & Trapero, A. (2005b). El chancro carbonoso de Quercus III: Dispersión de ascosporas del agente causal. Boletín de sanidad vegetal. Plagas, 31(4), 577–585.Google Scholar
  18. La Porta, N., Capretti, P., Thomsen, I. M., Kasanen, R., Hietala, A. M., & Von Weissenberg, K. (2008). Forest pathogens with higher damage potential due to climate change in Europe. Canadian Journal of Plant Pathology, 30(2), 177–195.CrossRefGoogle Scholar
  19. Mazzaglia, A., Anselmi, N., Gasbarri, A., & Vannini, A. (2001a). Development of Polymerase Chain Reaction (PCR) assay for the specific detection of Biscogniauxia mediterranea living as an endophyte in oak tissues. Mycological Research, 105(8), 952–956.CrossRefGoogle Scholar
  20. Mazzaglia, A., Anselmi, N., Vicario, S., & Vannini, A. (2001b). Sequence analysis of the 5.8S and ITS regions in evaluating genetic relationships among some species of Hypoxylon and related genera. Mycological Research, 105(6), 670–675.CrossRefGoogle Scholar
  21. Papke, R. T., & Ward, D. M. (2004). The importance of physical isolation to microbial diversification. FEMS Microbiology Ecology, 48(3), 293–303.CrossRefPubMedGoogle Scholar
  22. Peakall, R., & Smouse, P. E. (2006). GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes, 6(1), 288–295.CrossRefGoogle Scholar
  23. Peakall, R., & Smouse, P. E. (2012). GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research – an update. Bioinformatics, 28(19), 2537–2539.PubMedCentralCrossRefPubMedGoogle Scholar
  24. Peláez, F., González, V., Platas, G., Sánchez-Ballesteros, J., & Rubio, V. (2008). Molecular phylogenetic studies within the Xylariaceae based on ribosomal DNA sequences. Fungal Diversity, 31, 111–134.Google Scholar
  25. Santos, M. N. S. (2003). Contribuição para o conhecimento das relações Quercus suber- Biscogniauxia mediterranea (syn. Hypoxylon mediterraneum). Silva Lusitana, 11(1), 21–29.Google Scholar
  26. Schiaffino, A., Francheschini, A., Maddau, L., & Serra, S. (2002). Molecular characterization of Biscogniauxia mediterranea (De Not.) strains isolated from the declining trees of Quercus suber L. in Sardania. IOBC/wprs Bulletin, 25(5), 37–40.Google Scholar
  27. Sousa, E., Santos, M. N., Varela, M. C., & Henriques, J. (2007). Perda do vigor dos montados de sobro e azinho: análise da situação e perspetivas (Documento síntese). MADRP; DGRF; INRB, I.P.Google Scholar
  28. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30(12), 2725–2729.PubMedCentralCrossRefPubMedGoogle Scholar
  29. Tang, A. M. C., Jeewon, R., & Hyde, K. D. (2009). A re-evaluation of the evolutionary relationships within the Xylariaceae based on ribosomal and protein-coding gene sequences. Fungal Diversity, 34, 155–153.Google Scholar
  30. Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673–4680.PubMedCentralCrossRefPubMedGoogle Scholar
  31. Uddin, W., & Stevenson, K. L. (1997). Pathogenicity of a species of Phomopsis causing a shoot blight on peach in Georgia and evaluation of possible infection courts. Plant Disease, 81(9), 983–989.CrossRefGoogle Scholar
  32. Vannini, A., Mazzaglia, A., & Anselmi, N. (1999). Use of random amplified polymorphic DNA (RAPD) for detection of genetic variation and proof of the heterothallic mating system in Hypoxylon mediterraneum. European Journal of Plant Pathology, 29(3), 209–218.CrossRefGoogle Scholar
  33. White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR Protocols: a guide to methods and applications (pp. 315–322). San Diego: Academic Press.Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Joana Henriques
    • 1
    Email author
  • Filomena Nóbrega
    • 1
  • Edmundo Sousa
    • 1
  • Arlindo Lima
    • 2
  1. 1.Instituto Nacional de Investigação Agrária e Veterinária, I.P. – Unidade Estratégica de Sistemas Agrários e Florestais e Sanidade VegetalOeirasPortugal
  2. 2.Centro de Engenharia dos Biossistemas, Instituto Superior de AgronomiaUniversidade de LisboaLisboaPortugal

Personalised recommendations