1.8.37
DEVELOPMENT OF MONOCLONAL ANTIBODY-BASED IMMUNOASSAY TO DETERMINE BIOMASS OF CLADOSPORIUM FULVUM IN INFECTED TOMATO LEAVES

N KARPOVICH-TATE, P SPANU and FM DEWEY

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK

Background and objectives
Few satisfactory methods have been developed which accurately determine the biomass of specific plant pathogenic fungi within host tissues [1], but almost no attempt has been made to compare and validate such methods. The aim of this project was to develop the monoclonal antibody-based immunoassay for quantification of Cladosporium fulvum in infected tomato leaves (Lycopersicum esculentum) at all stages of infection, and to validate this method with the biomass measurements using glucuronidase (GUS) activity in GUS-transformant strain of C. fulvum [2].

Results and conclusions
Monoclonal antibody, OX-CH1, was raised against surface washings of Cladosporium herbarum. This antibody recognizes an epitope which is found in fungi belonging to the genus Cladosporium, including C. fulvum, the causal agent of tomato leaf mould. The epitope is present at comparable levels in two different races of C. fulvum and in transgenic isolates derived from them. The epitope is heat- and protease-resistant, but sensitive to oxidation with periodate and is constitutively expressed in C. fulvum grown in pure culture and on the plant. C. fulvum can be detected in infected tissues at levels starting from around 1 mg g-1 fresh weight. Non-infected tomato leaves do not cross--react with OX-CH1. We have developed a plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) for determining the fungal biomass in tomato leaves, and compared it with the assay based on measurements of GUS activity in tissues infected with a GUS-carrying transgenic isolate of C. fulvum.

The two assays give remarkably similar results in regard to both sensitivity and the course of infection. The GUS assay is sensitive and easy to carry out, but is limited to fungal isolates that have been transformed with the bacterial GUS gene. In this respect the PTA assay is of greater general applicability, as it can be employed for a variety of different isolates of C. fulvum and does not require the use of transgenic organisms. This assay is robust and simple and will be useful in the analysis of the effect of directed mutations on the pathogenicity of the fungus, in identifying new pathogenicity genes in C. fulvum, and for following fungal biomass during infection in the interaction between different races of the pathogen in tomato plants carrying different resistance genes.

References
1. Dewey FM, Thornton CR, Gilligan CA, 1997. Advances in Botanical Research 24, 276-308.
2. Oliver RP, Farman ML, Jones JDG, Hammond-Kosack KE, 1993. Molecular Plant-Microbe Interactions 6, 521-25.