Abstract
Polyclonal antibodies were raised against mycelium from the logarithmic growth phase of a shake culture of Ustilago nuda, and a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated detection antibodies was developed. The detection limit of the assay was 15 ng total protein ml−1 for the homologous antigen and 50 ng ml−1 for a spore extract, respectively. Other species of Ustilago reacted with the antibodies. Cross-reactivity was highest with U. tritici. No signal was obtained with the tested isolates of Tilletia, Rhizoctonia, Pythium and Fusarium. With naturally infected barley seeds, the results of the ELISAs were always in good agreement with those obtained with the routinely used seed embryo test. However, when seeds grown from artificially inoculated florets were used, the ELISA indicated significantly higher infestation levels than the embryo test. Results of assays with halved seeds from the same lot showed that high amounts of mycelium were present in the non-embryo half. This and especially the relatively long duration of the assay suggested that the ELISA (as conducted here) may not be suitable as a routine method for analysing seed infection with U. nuda. With samples from barley seedlings grown from infected seeds the results of the immunoassay again corresponded very well with the infection level determined by staining of the seed embryo, irrespective of the mode of floret inoculation (natural or artificial). Potential fields of application of the ELISA include the early prediction of the efficacy of protection agents, e.g. in screenings for seed treatments, the elucidation of the biology of the fungus and characterisation of resistance mechanisms.
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Abbreviations
- cv.:
-
cultivar
- EC:
-
growth stage according to Zadoks et al., 1974
- IgG:
-
Immunoglobuline G
- ISTA:
-
International Seed Testing Association
- PBS:
-
phosphate buffered saline
References
GC Ainsworth K Sampson (1950) The British smut fungi (Ustilaginales) Commonwealth Mycological Institute, Kew UK
F Bieber (1990) Biotinylierung monoklonaler Antikörper JH Peters H Baumgarten (Eds) Monoklonale Antikörper: Herstellung und Charakterisierung Springer Verlag Berlin, Germany 299–302
MM Bradford (1976) ArticleTitleA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72 248–254 Occurrence Handle942051 Occurrence Handle1:CAS:528:DyaE28XksVehtrY%3D
LM Brill RD McClary JB Sinclair (1994) ArticleTitleAnalysis of two ELISA formats and antigen preparations using polyclonal antibodies against Phomopsis longicolla Phytopathology 84 173–179 Occurrence Handle1:CAS:528:DyaK2cXktVCgu7w%3D
R Černuško GA Wolf (1997) Immunological methods for the detection of Verticillium dahliae in oilseed rape H Dehne G Adam Diekmann M J Frahm A Mauler-Machnik P van Halteren (Eds) Diagnosis and Identification of Plant Pathogens. Proceedings of the 4th International Symposium of the European Foundation for Plant Pathology Kluwer Academic Publishers Dordrecht, The Netherlands
MF Clark (1981) ArticleTitleImmunosorbent assays in Plant Pathology Annual Review of Phytopathology 19 83–106 Occurrence Handle1:CAS:528:DyaL3MXls1Crsr8%3D
MF Clark AN Adams (1977) ArticleTitleCharacteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses Journal of general Virology 34 475–483 Occurrence Handle10.1099/0022-1317-34-3-475 Occurrence Handle323416 Occurrence Handle1:STN:280:DyaE2s7mslWntQ%3D%3D
FM Dewey CR Thornton (1995) Fungal immunodiagnostics in plant agriculture JH Skerritt R Appels (Eds) New Diagnostics in Crop Sciences CAB International Wallingford, UK 151–170
Eibel P (2002) Entwicklung und Erprobung immunologischer und molekulargenetischer Methoden zur Frühdiagnose von Ustilago nuda (Jens.) Rostrup und Tilletia caries (D. C.) Tul. & C. Tul. in Gerste und Weizen. Der Andere Verlag, Osnabrück, Germany (doctoral thesis at the Department of Agricultural Sciences, Georg August University, Göttingen, Germany)
GW Fischer CS Holton (1957) Biology and Control of the Smut Fungi The Ronald Press Company New York, USA
GW Fischer CG Shaw (1953) ArticleTitleA proposed species concept in the smut fungi, with application to North American species Phytopathology 43 181–188
RW Goth PJ Ellis Villiers G de EW Goins NS Wright (1999) ArticleTitleCharacteristics and Distribution of Potato Latent Carlavirus (Red LaSoda Virus) in North America Plant Disease 83 751–753
K Goulter J Randles (1997) Serological and molecular techniques to detect and identify plant pathogens JF Brown HJ Ogle (Eds) Plant pathogens and plant diseases Rockvale Publications Armidale, Australia 172–191
WK Kim R Rohringer J Nielsen (1984) ArticleTitleComparison of polypeptides in Ustilago spp. pathogenic on wheat, barley, and oats: A chemotaxonomic study Canadian Journal of Botany 62 1431–1437 Occurrence Handle1:CAS:528:DyaL2cXlt1KntLk%3D
B Lindeberg JA Nannfeldt (1959) ArticleTitleUstilaginales of Sweden Symbola Botanicae Upsaliensis 16 1–175
MMS Malik CCV Batts (1960a) ArticleTitleThe infection of barley by loose smut (Ustilago nuda (Jens) Rostr) Transactions of the British Mycological Society 43 117–125
MMS Malik CCV Batts (1960b) ArticleTitleThe development of loose smut of barley (Ustilago nuda) in the barley plant, with observations on spore formation in nature and in culture Transactions of the British Mycological Society 43 126–131 Occurrence Handle10.1016/S0007-1536(60)80016-2
JG Menzies G Bakkeren F Matheson JD Procunier S Woods (2003) ArticleTitleUse of inter-simple sequence repeats and amplified fragment length polymorphisms to analyze genetic relationships among small grain-infecting species of Ustilago Phytopathology 93 167–175 Occurrence Handle1:CAS:528:DC%2BD3sXhtFGhsLg%3D Occurrence Handle18943131
UM Meyer RA Spotts FM Dewey (2000) ArticleTitleDetection and quantification of Botrytis cinera by ELISA in pear stems during cold storage Plant Disease 84 1099–1103 Occurrence Handle1:CAS:528:DC%2BD3cXntFOgsrk%3D
SB Mohan (1988) ArticleTitleEvaluation of antisera raised against Phytophtora fragariae for detecting the red core disease of strawberries by enzyme-linked immmunosorbent assay (ELISA) Plant Pathology 37 206–216
MB Moore (1936) ArticleTitleA method for inoculating wheat and barley with loose smut Phytopathology 26 397–400
DJ Morton (1961) ArticleTitleTrypan blue and boiling lactophenol for staining and clearing barley tissues infected with Ustilago nuda Phytopathology 51 27–29 Occurrence Handle1:CAS:528:DyaF3MXntFaktw%3D%3D
J Nielsen (1972) ArticleTitleIsolation and culture of monokaryotic haplonts of Ustilago tritici, observations on their physiology, and the taxonomic relationship between U tritici and U. nuda. Canadian Journal of Botany 50 1775–1781 Occurrence Handle10.1139/b72-221
KL Perry FL Kolb B Sammons C Lawson G Cisar H Ohm (2000) ArticleTitleYield Effects of Barley yellow dwarf virus in Soft Red Winter Wheat Phytopathology 90 1043–1048 Occurrence Handle18944532 Occurrence Handle1:STN:280:DC%2BD1cjjslyhuw%3D%3D
JM Poehlman (1945) ArticleTitleA simple method of inoculating barley with loose smut Phytopathology 35 640–644
Rennie WJ (1990) Working Sheet No. 25. In: ISTA Handbook on Seed Health Testing (Section 2: Working Sheets), International Seed testing Association, Zurich, Switzerland
WJ Rennie RD Seaton (1975) ArticleTitleLoose smut of barley: The embryo test as a means of assessing loose smut infection in seed stocks Seed Science and Technology 3 697–709
Schots A, Dewey FM and Oliver R (eds) (1994) Modern assays for plant pathogenic fungi: Identification, detection and quantification. CAB International, Wallingford, UK
CL Sutula JM Gillett SM Morrissey DC Ramsdell (1986) ArticleTitleInterpreting ELISA data and establishing the positive-negative threshold Plant Disease 70 722–726
CR Thornton T O’Neill G Hilton CA Gilligan (1999) ArticleTitleDetection and recovery of Rhizoctonia solani in naturally infested glasshouse soils using a combined baiting, double monoclonal antibody ELISA Plant Pathology 48 627–634
F Waldow (1997) Entwicklung und Erprobung immunologischer Methoden zum Nachweis des Kohlhernieerregers Plasmodiophora brassicae (Woron.) für Phytohygieneprüfungen von Komposten Cuvillier Verlag Göttingen, Germany
JC Zadoks TT Chang CF Konzak (1974) ArticleTitleA decimal code for the growth stage of cereals Weed Research 14 415–421
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Eibel, P., Wolf, G.A. & Koch, E. Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of loose smut of barley (Ustilago nuda). Eur J Plant Pathol 111, 113–124 (2005). https://doi.org/10.1007/s10658-004-1421-z
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DOI: https://doi.org/10.1007/s10658-004-1421-z