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Differential interactions of Verticillium longisporum and V. dahliae with Brassica napus detected with molecular and histological techniques

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Abstract

The differential interactions of V. longisporum (VL) and V. dahliae (VD) on the root surface and in the root and shoot vascular system of Brassica napus were studied by confocal laser scanning microscopy (CLSM), using GFP tagging and conventional fluorescence dyes, acid fuchsin and acridin orange. VL and VD transformants expressing sGFP were generated by Agrobacterium-mediated transformation. GFP signals were less homogenous and GFP tagging performed less satisfactory than the conventional fluorescence staining when both were studied with CLSM. Interactions of both pathogens were largely restricted to the root hair zone. At 24 h post-inoculation (hpi), hyphae of VL and VD were found intensely interwoven with the root hairs. Hyphae of VL followed the root hairs towards the root surface. At 36 hpi, VL hyphae started to cover the roots with a hyphal net strictly following the grooves of the junctions of the epidermal cells. VL started to penetrate the root epidermal cells without any conspicuous infection structures. Subsequently, hyphae grew intracellularly and intercellularly through the root cortex towards the central cylinder, without inducing any visible plant responses. Colonisation of the xylem vessels in the shoot with VL was restricted to individual vessels entirely filled with mycelium and conidia, while adjacent vessels remained completely unaffected. This may explain why no wilt symptoms occur in B. napus infected with VL. Elevated amounts of fungal DNA were detectable in the hypocotyls 14 days post-inoculation (dpi) and in the leaves 35 dpi. Root penetration was also observed for VD, however, with no directed root surface growth and mainly an intercellular invasion of the root tissue. In contrast to VL, VD started ample formation of conidia on the roots, and was unable to spread systemically into the shoots. VD did not form microsclerotia in the root tissue as widely observed for VL. This study confirms that VD is non-pathogenic on B. napus and demonstrates that non-host resistance against this fungus materializes in restriction of systemic spread rather than inhibition of penetration.

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References

  • Babadoost, M., Chen, W., Bratsch, A. D., & Eastman, C. E. (2004). Verticillium longisporum and Fusarium solani: two new species in the complex of internal discolouration of horseradish roots. Plant Pathology, 53, 669–676.

    Article  Google Scholar 

  • Beckmann, C. H. (1987). The nature of wilt diseases of plants. St. Paul, MN, USA: APS Press.

    Google Scholar 

  • Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research, 12, 8711–8721.

    Article  PubMed  CAS  Google Scholar 

  • Bhat, R. G., & Subbarao, K. V. (1999). Host range specificity in Verticillium dahliae. Phytopathology, 89, 1218–1225.

    Article  PubMed  CAS  Google Scholar 

  • Bolwerk, A., Lagopodi, A., Lugtenberg, B. J. J., & Bloemberg, G. V. (2005). Visualization of interactions between a pathogenic and a benefical Fusarium strain during biocontrol of tomato foot and root rot. Molecular Plant-Microbe Interactions, 18, 710–721.

    Article  PubMed  CAS  Google Scholar 

  • Buckley, P. M., Wyllie, T. D., & DeVay, J. E. (1969). Fine structure of conidia and conidium formation in Verticillium albo-atrum and V. nigrescens. Mycologia, 61, 240–250.

    Article  Google Scholar 

  • Bundock, P., den Dulk-Ras, A., Beijersbergen, A., & Hoykaas, P. J. J. (1995). Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. European Molecular Biology Organization, 14, 3206–3214.

    CAS  Google Scholar 

  • Campbell, C. L., & Madden, L. V. (1990). Introduction to plant disease epidemiology. New York, USA: John Wiley.

    Google Scholar 

  • Chalfie, M., & Kain, S. (1998). Green fluorescent protein. properties, applications and protocols. New York: Wiley-Liss, Inc.

    Google Scholar 

  • Collins, A., Okoli, C. A. N., Morton, A., Parry, D., Edwards, S. G., & Barbara, D. J. (2003). Isolates of Verticillium dahliae pathogenic to crucifers are of at least three distinct molecular types. Phytopathology, 93, 364–376.

    Article  PubMed  Google Scholar 

  • Covert, S. F., Kapoor, P., Lee, M., Briley, A., & Nairn, C. J. (2001). Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycological Research, 105, 259–264.

    Article  CAS  Google Scholar 

  • Daebeler, F., Amelung, D., & Zeise, K. (1988). Verticillium-Welke an Winterraps—Auftreten und Bedeutung. Nachrichtenblatt Pflanzenschutzdienst DDR, 42, 71–73.

    Google Scholar 

  • Dimond, A. E. (1970). Biophysics and biochemistry of the vascular wilt syndrome. Annual Review of Phytopathology, 8, 301–322.

    Article  Google Scholar 

  • Dixelius, C., Happstadius, I., & Berg, G. (2005). Verticillium wilt on Brassica oil crops—a Swedish perspective. Journal of the Swedish Seed Association, 115, 36–48.

    Google Scholar 

  • Dixon, G. R., & Pegg, G. F. (1972). Changes in the amino acid content of tomato xylem sap following infection with strains of Verticillium albo-atrum. Annals of Botany, 36, 147–154.

    CAS  Google Scholar 

  • Dobinson, K. F. (1994). Genetic transformation of the vascular wilt fungus Verticillium dahliae. Canadian Journal of Botany, 73, 710–715.

    Google Scholar 

  • Domsch, K. H., Gams, W., & Anderson, T. -H. (1980). Nectria (Fr) 1849, Verticillium Nees ex Link 1824. In: Compendium of Soil Fungi (pp. 829–845), Vol 1. New York: Academic Press.

  • Eckert, M., Maguire, K., Urban, M., Foster, S., Fitt, B., Lucas, J., & Hammond-Kosack, K. (2005). Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp. FEMS Microbiology Letters, 253, 67–74.

    Article  PubMed  CAS  Google Scholar 

  • Fahleson, J., Lagercrantz, U., Hu, Q., Steventon, L. A., & Dixelius, C. (2003). Estimation of genetic variation among Verticillium isolates using AFLP analysis. European Journal of Plant Pathology, 109, 361–371.

    Article  CAS  Google Scholar 

  • Gold, J., Lee, B., & Robb, J. (1996). Colonization of tomatoes by Verticillium dahliae: determinative phase II. Canadian Journal of Botany, 74, 1279–1288.

    Article  Google Scholar 

  • Green, R. J. J. (1981). An overview. In M. E. Mace, A. A. Bell, & C. H. Beckman (Eds.), Fungal wilt diseases of plants (pp. 1–24). New York: Academic Press.

    Google Scholar 

  • Günzelmann, H., & Paul, V. H. (1990). Zum Auftreten und zur Bedeutung der Verticillium-Welke an Raps in der Bundesrepublik Deutschland in 1989. Raps, 8, 23–25.

    Google Scholar 

  • Hanahan, J. (1983). Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology, 166, 557–580.

    Article  PubMed  CAS  Google Scholar 

  • Happstadius, I., Ljunberg, A., Kristiansson, B., & Dixelius, C. (2003). Identification of Brassica oleracea germplasm with improved resistance to Verticillium wilt. Plant Breeding, 122, 30–34.

    Article  Google Scholar 

  • Heale, J. B., & Karapapa, V. K. (1999). The Verticillium threat to Canada`s major oilseed crop: Canola. Canadian Journal of Plant Pathology, 21, 1–7.

    Google Scholar 

  • Heinz, R., Lee, S. W., Saparno, A., Nazar, R. N., & Robb, J. (1998). Cyclical systemic colonization in Verticillium-infected tomato. Physiological and Molecular Plant Pathology, 52, 385–396.

    Article  Google Scholar 

  • Hood, E. E., Helmer, G. L., Fraley, R. T., & Chilton, M. D. (1986). The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. Journal of Bacteriology, 168, 1291–1301.

    PubMed  CAS  Google Scholar 

  • Horowitz, S., Freeman, S., & Sharon, A. (2002). Use of green fluorescent protein-transgenic strains to study pathogenic and nonpathogenic lifestyles in Colletotrichum acutatum. Phytopathology, 92, 743–749.

    Article  PubMed  Google Scholar 

  • Johannson, A., Goud, J. -K., & Dixelius, C. (2006). Plant host range of Verticillium longisporum and microsclerotia density in Swedish soils. European Journal of Plant Pathology, 114, 139–149.

    Article  Google Scholar 

  • Karapapa, V. K., Baig, M. A., Heale, J. B., & Rossiter, J. T. (1997a). Glucosinolate response in winter oilseed rape Brassica napus ssp. oleifera to Verticillium dahliae (non-pathogenic), V. longisporum comb. Nov., (Karapapa, Bainbridge and Heale, 1997) (pathogenic). In E. C. Tjamos, R. C. Rowe, J. B. Heale, & D. R. Fravel (Eds.), Advances in Verticillium research and disease management. St. Paul, Minnesota: APS Press.

    Google Scholar 

  • Karapapa, V. K., Bainbridge, B. W., & Heale, J. B. (1997b). Morphological and molecular characterisation of Verticillium longisporum comb. nov., pathogenic to oilseed rape. Mycological Research, 101, 1281–1294.

    Article  Google Scholar 

  • Komari, T., Halperin, W., & Nester, E. W. (1986). Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542. Journal of Bacteriology, 166, 88–94.

    PubMed  CAS  Google Scholar 

  • Krüger, W. (1989). Untersuchungen zur Verbreitung von Verticillium dahliae Kleb. und anderen Krankheits- und Schaderregern bei Raps in der Bundesrepublik Deutschland. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes, 41, 49–56.

    Google Scholar 

  • Lagopodi, A. L., Ram, A. F. J., Lamers, G. E. M., & Punt, P. J. (2001). Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker. Molecular Plant-Microbe Interactions, 15, 172–179.

    Article  Google Scholar 

  • Lazo, G. R., Stein, P. A., & Ludwig, R. A. (1991). A DNA transformation–competent Arabidopsis genomic library in Agrobacterium. Bio/Technology, 9, 963–967.

    Article  PubMed  CAS  Google Scholar 

  • Lorang, J. M., Tuori, R. P., Martinez, J. P., Sawyer, T. L., Redman, R. S., Rollins, J. A., Wolpert, T. J., Johnson, K. B., Rodriguez, R. J., Dickman, M. B., Ciufetti, L. M. (2001). Green fluorescent protein is lighting up fungal biology. Applied and Environmental Microbiology, 67, 1987–1994.

    Article  PubMed  CAS  Google Scholar 

  • Maor, R., Puyesky, M., Horwitz, B. A., & Sharon, A. (1998). Use of green fluorescent protein (GFP) for studying development and fungal-plant interaction in Cochliobolus heterostrophus. Mycological Research, 102, 491–496.

    Article  Google Scholar 

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

  • Melouk, H. (1992). Verticillium. In L. L. Singleton, J. D. Mihail, & C. M. Rush (Eds.), Methods for research on soilborne pathogenic fungi (pp. 175–178). St Paul, MN, USA: APS Press.

    Google Scholar 

  • Mol, L., & Scholte, K. (1995). Formation of microsclerotia of Verticillium dahliae Kleb. on various plant parts of two potato cultivars. Potato Research, 38, 143–150.

    Article  Google Scholar 

  • Neumann, M. J., & Dobinson, K. F. (2003). Sequence tag analysis of gene expression during pathogenic growth and microsclerotia development in the vascular wilt pathogen Verticillium dahliae. Fungal Genetics and Biology, 38, 54–62.

    Article  PubMed  CAS  Google Scholar 

  • Oren, L., Ezrati, S., Cohen, D., & Sharon, A. (2003). Early events in the Fusarium verticillioides-maize interaction characterized by using a green fluorescent protein-expressing transgenic isolate. Applied and Environmental Microbiology, 69, 1695–1701.

    Article  PubMed  CAS  Google Scholar 

  • Pegg, G. F. (1981). Biochemistry and physiology of pathogenesis. In M. E. Mace, A. A. Bell, & C. H. Beckman (Eds.), Fungal wilt diseases of plants (pp. 193–253). New York: Academic Press.

    Google Scholar 

  • Pegg, G. F. (1984). The impact of Verticillium diseases in agriculture. Phytopathologia Mediterranea, 23, 176–192.

    Google Scholar 

  • Pegg, G. F. (1985). Life in a black hole—the micro-environment of the vascular pathogen. Transactions of the British Mycological Society, 85, 1–20.

    Article  Google Scholar 

  • Pegg G. F., & Brady B. L. (Eds.) (2002). Hosts. In: Verticillium Wilts (pp. 193–340). Wallingford, UK: CAB Publishing.

  • Punt, P. J., Oliver, R., Dingemanse, M. A., Pouwels, P. H., & van den Hondel, C. A. M. J. J. (1987). Transformation of Aspergillus based on the Hygromycin B resistance marker from Escherichia coli. Gene, 56, 117–124.

    Article  PubMed  CAS  Google Scholar 

  • Robinson, M., & Sharon, A. (1999). Transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia. Current Genetics, 36, 98–104.

    Article  PubMed  CAS  Google Scholar 

  • Rowe, R. R., & Powelson, M. L. (2002). Potato early dying: management challenges in a changing production environment. Plant Diseases, 86, 1184–1193.

    Article  Google Scholar 

  • Schnathorst, W. C. (1981). Life cycle and epidemiology of Verticillium. In M. E. Mace, A. A. Bell, & C. H. Beckmann (Eds.), Fungal wilt diseases of plants (pp. 81–111). New York: Academic Press.

    Google Scholar 

  • Shan, X. C., & Goodwin, P. H. (2004). Monitoring host nuclear migration and degradation with green fluorescent protein during compatible and incompatible interactions of Nicotiana tabacum with Colletotrichum species. Journal of Phytopathology, 152, 454–560.

    Article  CAS  Google Scholar 

  • Short, J. M., Fernandez, J. M., Sorge, J. A., & Huse, W. D. (1988). Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acid Research, 16, 7583–7600.

    Article  CAS  Google Scholar 

  • Söchting, H. P., & Verreet, J. -A. (2004). Effects of different cultivation systems (soil management, nitrogen fertilization) on the epidemics of fungal diseases in oilseed rape (Brassica napus L. var. napus). Journal of Plant Diseases and Protection, 111, 1–29.

    Google Scholar 

  • Spellig, T., Bottin, A., & Kahmann, R. (1996). Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis. Molecular and General Genetics, 252, 503–509.

    PubMed  CAS  Google Scholar 

  • Stark, C. (1961). Das Auftreten der Verticillium-Tracheomykosen in Hamburger Gartenbau-Kulturen. Gartenbauwissenschaft, 26, 493–528.

    Google Scholar 

  • Subbarao, K. V., Chassot, A., Gordon, T. R., Hubbard, J. C., Bonello, P., Mulin, R., Okamoto, D., Davis, R. M., & Koike, S. T. (1995). Host range of Verticillium dahliae from cauliflower and genetic relationships and cross pathogenicities of isolates from different crops. Phytopathology, 85, 1105–1112.

    Article  Google Scholar 

  • Svenson, C. H., & Lerenius, C. (1987). An investigation on the effect of Verticillium wilt (Verticillium dahliae Kleb.) on oilseed rape. Working group integrated control in oilseed rape. IOBC/WPRS Bulletin, X/4, 30–34.

    Google Scholar 

  • Tsien, R. Y. (1998). The green fluorescent protein. Annual Review of Biochemistry, 67, 509–544.

    Article  PubMed  CAS  Google Scholar 

  • Van Alfen, N. K. (1989). Molecular bases for virulence and avirulence of fungal wilt pathogens. In E. C. Tjamos & C. H. Beckman (Eds.), Vascular wilt diseases of plants (pp. 19–32). Berlin: Springer.

    Google Scholar 

  • Wen-Jun, S., & Forde, B. G. (1989). Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acid Research, 17, 8385.

    Article  CAS  Google Scholar 

  • Wood, R. K. S. (1961). Verticillium wilt of tomatoes and the role of pectic and cellulolytic enzymes. Annals of Applied Biology, 49, 120–139.

    Article  CAS  Google Scholar 

  • Xiao, C. L., & Subbarao, K. V. (2000). Effects of Irrigation and Verticillium dahliae on cauliflower root and shoot growth dynamics. Phytopathology, 90, 995–1004.

    Article  PubMed  CAS  Google Scholar 

  • Zeise, K., & Seidel, D. (1990). Zur Entwicklung und Schadwirkung der Verticillium- Welkekrankheit am Winterraps. Raps, 8, 20–22.

    Google Scholar 

  • Zeise, K. (1992). Gewächshaustest zur Resistenzprüfung von Winterraps (Brassica napus L. var. oleifera Metzger) gegen den Erreger der Rapswelke Verticillium dahliae Kleb. Nachrichtenblatt Deutscher Pflanzenschutzdienst, 44, 125–128.

    Google Scholar 

  • Zeise, K., & von Tiedemann, A. (2001). Morphological and physiological differentiation among vegetative compatibility groups of Verticillium dahliae in relation to V. longisporum. Journal of Phytopathology, 149, 469–475.

    Article  Google Scholar 

  • Zeise, K., & von Tiedemann, A. (2002a). Application of RAPD-PCR for virulence type analysis within Verticillium dahliae and Verticillium longisporum. Journal of Phytopathology, 150, 557–563.

    Article  CAS  Google Scholar 

  • Zeise, K., & von Tiedemann, A. (2002b). Host specialization among vegetative compatibility groups of Verticillium dahliae in relation to Verticillium longisporum. Journal of Phytopathology, 150, 112–119.

    Article  Google Scholar 

  • Zhou, L., Hu, Q., Johannson, A., & Dixelius, C. (2006). Verticillium longisporum and Verticillium dahliae: Infection and disease in Brassica napus. Plant Pathology, 55, 137–144.

    Article  CAS  Google Scholar 

  • Zielenski, D., & Sadowski, C. (1995). A preliminary study on Verticillium dahliae Kleb. in winter oilseed rape in Poland. In D. Murphy (Ed.), Proceedings of the 9th International Rapeseed Conference, Cambridge. 4–7 July 1995. GciRC, Cambridge, UK, 649–651.

  • Zou, W. J., Yoneyama, K., Takeuchi, Y., Iso, S., Rugmekarat, S., Chae, S. H., Sato, D., & Joel, D. M. (2004). In vitro infection of host roots by differentiated calli of the parasitic plant Orobranche. Journal of Experimental Botany, 55, 899–907.

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to the breeding companies represented by the GFP (Gemeinschaft zur Förderung der privaten deutschen Pflanzenzüchtung e.V.) for constant support and fruitful cooperation. The funding of this study by GFP and FNR (German Ministry of Food, Agriculture and Consumer Protection) is acknowledged.

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Eynck, C., Koopmann, B., Grunewaldt-Stoecker, G. et al. Differential interactions of Verticillium longisporum and V. dahliae with Brassica napus detected with molecular and histological techniques. Eur J Plant Pathol 118, 259–274 (2007). https://doi.org/10.1007/s10658-007-9144-6

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