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Quantification of the effect of the hyperparasite Trichoderma harzianum on the saprotrophic growth dynamics of Rhizoctonia solani in compost using a monoclonal antibody-based ELISA

Published online by Cambridge University Press:  01 April 1999

C. R. THORNTON
Affiliation:
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K. Current address: Department of Biological Sciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS, U.K.
C. A. GILLIGAN
Affiliation:
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
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Abstract

This paper describes the use of a monoclonal antibody-based ELISA for the quantification of the effects of Trichoderma harzianum on the saprotrophic growth dynamics of Rhizoctonia solani in unsterile compost-based microcosms. Quantification of R. solani was based on the immunological detection of a constitutive, extracellular, water soluble glycoprotein antigen (the copper-containing enzyme catechol oxidase) which is secreted by actively growing mycelium of the fungus only. Extracts derived from lyophilized mycelium (FDM) were used as a quantifiable and repeatable source of the antigen in order to prepare calibration curves that accounted for incomplete extraction of the antigen from compost samples. These curves were used to convert the absorbance values of extracts in ELISA to FDM biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of R. solani to be made in the presence and absence of T. harzianum. The high pH (9·6) of the buffer used to extract the antigen from microcosm samples also resulted in the extraction of humic components of the compost which interfered with the sensitivity of the assay. A protocol, based on the determination of optimal ELISA absorbance[ratio ]extract dilution ratios, was used in order to reduce the interference and improve the sensitivity of the quantitative assay.

Type
Research Article
Copyright
The British Mycological Society 1999

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