SpringerLink
Log in

Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus

  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in bulbs in storage. In order to study the mechanisms of pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B phosphotransferase (hph) gene and fluorescence reporter genes EGFP (green), EYFP (yellow) or ECFP (cyan) using the AGL-1 strain of A. tumefaciens. Hygromycin B (100 μg/ml) resistant colonies were observed only when acetosyringone was added to the co-cultivation medium. Transformed colonies are more clearly visible when co-cultivated on cellophane membrane than on Hybond -N+ membrane. Transformed lines were stably maintained through four serial passages on medium containing hygromycin B, and they expressed green, yellow or cyano fluorescence. PCR with hph-specific primers and Southern blotting with an hph-specific probe were positive for HygR lines but not for the untransformed isolate. The cyano fluorescence of the ECFP-transformed isolate was clearly distinguishable from the green autofluorescence of Gladiolus roots, signifying the potential of these lines for further histopathological investigations. Transformed lines will be useful for identifying pathogenicity related genes, screening transgenic resistance, and in studies of host-pathogen interactions.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

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

References

  • Agrios, G. N. (2005). Plant pathology (5th ed.). London: Elsevier Academic Press.

    Google Scholar 

  • Chen, X., Stone, M., Schlagnhaufer, C., & Romaine, C. P. (2000). A fruiting body tissue method for efficient agrobacterium-mediated transformation of Agaricus bisporus. Applied and Environmental Microbiology, 66(10), 4510–4513.

    Article  PubMed  CAS  Google Scholar 

  • Cubero, O. F., Crespo, A., Fatehi, J., & Bridge, P. D. (1999). DNA extraction and PCR amplification method suitable for fresh, herbarium and lichenized fungi. Plant Systematics and Evolution, 216(3), 243–249.

    Article  CAS  Google Scholar 

  • Degefu, Y., & Hanif, M. (2003). Agrobacterium tumefaciens-mediated transformation of Helminthosporium turcicum, the maize leafblight fungus. Archives of Microbiology, 180(4), 279–284.

    Article  PubMed  CAS  Google Scholar 

  • Dhingra, O. D., & Sinclair, J. B. (1995). Basic plant pathology methods (2nd ed.). Boca Raton: CRC/Lewis.

    Google Scholar 

  • Elmer, W. H. (2006). Efficacy of preplant treatments of gladiolus corms with combinations of acibenzolar-S-methyl and biological or chemical fungicides for suppression of fusarium corm rot [Fusarium oxysporum f. sp. gladioli]. Canadian Journal of Plant Pathology, 28(4), 609–614.

    Article  CAS  Google Scholar 

  • Grimaldi, B., de Raaf, M. A., Filetici, P., Ottonello, S., & Ballario, P. (2005). Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards true genetics. Current Genetics, 48(1), 67–74.

    Article  Google Scholar 

  • Hanif, M., Pardo, A., Gorfer, M., & Raudaskoski, M. (2002). T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker. Current Genetics, 41(3), 183–188.

    Article  PubMed  CAS  Google Scholar 

  • Heimann, M. F., & Worf, G. L. (1997). Gladiolus disorder: Fusarium yellows and bulb rot. WI, USA: Cooperative Extension, University of Wisconsin, University Press. 274 pp.

    Google Scholar 

  • Jones, R. K., & Jenkins, J. M. (1975). Evaluation of resistance in Gladiolus sp. to Fusarium oxysporum f. sp. gladioli. Phytopathology, 65(4), 481–484.

    Article  Google Scholar 

  • Kemppainen, M., Circosta, A., Tagu, D., Martin, F., & Pardo, A. G. (2005). Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N. Mycorrhiza, 16(1), 19–22.

    Article  PubMed  CAS  Google Scholar 

  • Khan, M. R., & Mustafa, U. (2005). Corm rot and yellows of Gladiolus and its biomanagement. Phytopathologia Mediterranea, 44(2), 208–215.

    Google Scholar 

  • Khang, C., Park, S., Rho, H., Lee, Y., & Kang, S. (2006). Agrobacterium tumefaciens-mediated transformation and mutagenesis of filamentous fungi Magnaporthe grisea and Fusarium oxysporum. In K. Wang (Ed.), Agrobacterium protocols (pp. 403–420). Totowa: Humana.

    Chapter  Google Scholar 

  • Kilaru, S., Collins, C. M., Hartley, A. J., Bailey, A. M., & Foster, G. D. (2009). Establishing molecular tools for genetic manipulation of the pleuromutilin producing fungus Clitopilus passeckerianus. Applied and Environmental Microbiology, 75(22), 7196–7204.

    Article  PubMed  CAS  Google Scholar 

  • Kim, H.-S., Park, S.-Y., Lee, S., Adams, E. L., Czymmek, K., & Kang, S. (2011). Loss of cAMP-dependent protein kinase A affects multiple traits important for root pathogenesis by Fusarium oxysporum. Molecular Plant-Microbe Interactions, 24(6), 719–732.

    Article  PubMed  CAS  Google Scholar 

  • Lagopodi, A. L., Ram, A. F. J., Lamers, G. E. M., Punt, P. J., Van den Hondel, C. A. M. J. J., Lugtenberg, B. J. J., & Bloemberg, G. V. (2002). Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicislycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker. Molecular Plant-Microbe Interactions, 15(2), 172–179.

    Article  PubMed  CAS  Google Scholar 

  • Lakshman, D., Pandey, R., Kamo, K., Mitra, A., & Bauchan, G. (2010). Agrobacterium-mediated transformation of Fusarium oxysporum fsp. gladioli to study pathogenesis in Gladiolus. Phytopathology, 100(6).

  • Lievens, B., Rep, M., & Thomma, B. P. H. J. (2008). Recent developments in the molecular discrimination of formae speciales of Fusarium oxysporum. Pest Management Science, 64(8), 781–788.

    Article  PubMed  CAS  Google Scholar 

  • Löffler, H. J. M., Straathof, T. P., Van Rubroek, P. C. L., & Roebroeck, E. J. A. (1997). Fusarium resistance in gladiolus: the development of a screening assay. Journal of Phytopathology, 145(11–12), 465–468.

    Article  Google Scholar 

  • Magie, R. O. (1980). Fusarium diseases of gladioli controlled by inoculation of corms with non-pathogenic Fusaria. Proceedings of the Florida State Horticultural Society, 93, 172–175.

    Google Scholar 

  • Magie, R. O. (2000). Some Fungi That Attack Gladioli. Farming and Agriculture Series, Library4Farming. (http://science-in-farming.library4farming.org/index.html).

  • Maniatis, T., Fritsch, E. F., & Sambrook, J. (1982). Molecular cloning: A laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  • McDonnell, K. (1962). Relationship of pectic enzymes and pathogenicity in the fusarium wilt of tomatoes. Transactions of the British Mycological Society, 45(1), 55–62.

    Article  CAS  Google Scholar 

  • Michielse, C. B., & Rep, M. (2009). Pathogen profile update: Fusarium oxysporum. Molecular Plant Pathology, 10(3), 311–324.

    Article  PubMed  CAS  Google Scholar 

  • Michielse, C. B., van Wijk, R., Reijnen, L., Cornelissen, B. J. C., & Rep, M. (2009). Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis. GenomeBiology.com, 10(1), R4.

    PubMed  Google Scholar 

  • Mosquera, G., Giraldo, M. C., Khang, C. H., Coughlan, S., & Valent, B. (2009). Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as Biotrophy-associated secreted proteins in rice blast disease. The Plant Cell, 21(4), 1273–1290.

    Article  PubMed  CAS  Google Scholar 

  • Mullins, E., Chen, X., Romaine, P., Raina, R., Geiser, D., & Kang, S. (2001). Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 91(2), 173–180.

    Article  PubMed  CAS  Google Scholar 

  • Murashige, T., & Skoog, F. (1962). A revised medium for rapid assays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473–497.

    Article  CAS  Google Scholar 

  • Riaz, T., Khan, S. N., & Javaid, A. (2010). Screening of Gladiolus germplasm for agronomic performance and resistance against corm rot disease. African Journal of Biotechnology, 9(40), 6701–6707.

    Google Scholar 

  • Roebroeck, E. J. A., & Mes, J. J. (1992). Physiological races and vegetative compatibility groups within Fusarium oxysporum f.sp. gladioli. European Journal of Plant Pathology, 98(1), 57–64.

    Google Scholar 

  • Schenk, P. K., & Bergman, B. H. H. (1969). Uncommon disease symptoms caused by Fusarium oxysporum in tulips forced in the glasshouse after pre-cooling at 5°C. European Journal of Plant Pathology, 75, 100–104.

    Google Scholar 

  • Straathof, T. P., Roebroeck, E. J. A., & Löffler, H. J. M. (1998). Studies on Fusarium-Gladiolus interactions. Journal of Phytopathology, 146(2–3), 83–88.

    Article  Google Scholar 

  • Talhinhas, P., Muthumeenakshi, S., Neves-Martins, J., Oliveira, H., & Sreenivasaprasad, S. (2008). Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles. Molecular Biotechnology, 39(1), 57–67. doi:10.1007/s12033-007-9028-1.

    Article  PubMed  CAS  Google Scholar 

  • Thirugnanasambandam, A., Wright, K. M., Whisson, S. C., Atkins, S. D., & Newton, A. C. (2011). Infection of Rrs1 barley by an incompatible race of the fungus, Rhynchosporium secalis expressing the green fluorescent protein. Plant Pathology, 60(3), 513–521.

    Article  Google Scholar 

  • Tsuji, G., Fujihara, N., Hirose, C., Tsuge, S., Shiraishi, T., & Kubo, Y. (2003). Agrobacterium tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium. Journal of General Plant Pathology, 69(4), 230–239.

    Article  CAS  Google Scholar 

  • Ushimaru, T., Terada, H., Tsuboi, K., Kogou, Y., Sakaguchi, A., Tsuji, G., & Kubo, Y. (2010). Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer. Molecular Genetics and Genomics, 284(5), 357–371.

    Article  PubMed  CAS  Google Scholar 

  • Yang, Y., Cao, T., Yang, J., Howard, R. J., Kharbanda, P. D., & Strelkov, S. E. (2010). Histopathology of internal fruit rot of sweet pepper caused by Fusarium lactis. Canadian Journal of Plant Pathology, 32(1), 86–97.

    Article  CAS  Google Scholar 

  • Zhang, P., Xu, B., Wang, Y., Li, Y., Qian, Z., Tang, S., Huan, S., & Ren, S. (2008). Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei. Mycological Research, 112(8), 943–949.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Prof. Seogchan Kang (Penn State Univ., PA) for supplying the Agrobacterium strains containing the binary vectors, Prof. Robert McGovern (Univ. of Florida, Gainesville, FL) for supplying the F. oxysporum f.sp. gladioli used in this study, Siobhan O’Connor for the Southern analyses, and Chris Pooley (SGIL, USDA-ARS, Beltsville) for help with processing the confocal images.

Author information

Authors and Affiliations

  1. Floral and Nursery Plants Research Unit and Sustainable Agricultural Systems Lab, USDA-ARS, BARC, Beltsville, MD, 20705, USA

    Dilip K. Lakshman & Ruchi Pandey

  2. Floral and Nursery Plants Research Unit, USDA-ARS, BARC, Beltsville, MD, 20705, USA

    Kathryn Kamo

  3. Electron & Confocal Microscopy Unit, USDA-ARS, BARC, Beltsville, MD, 20705, USA

    Gary Bauchan

  4. Department of Plant Pathology, University of Nebraska, Lincoln, NE, 68583, USA

    Amitava Mitra

Authors
  1. Dilip K. Lakshman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ruchi Pandey
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kathryn Kamo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gary Bauchan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Amitava Mitra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dilip K. Lakshman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lakshman, D.K., Pandey, R., Kamo, K. et al. Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus . Eur J Plant Pathol 133, 729–738 (2012). https://doi.org/10.1007/s10658-012-9953-0

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-012-9953-0

Keywords

Search

Navigation