3.3.9
THE EFFECT OF METHOD OF ANTIGEN PREPARATION ON THE DETECTION OF FUSARIUM SPECIES BY MONOCLONAL ANTIBODIES

P JENNINGS, JA TURNER AND JN BANKS

Central Science Laboratory, Sand Hutton, York, YO4 1LZ.

Background and objectives
Disease symptoms caused by the different Fusarium species on wheat crops are often indistinguishable. Using conventional methods to identify the species responsible is time consuming and requires mycological expertise. Enzyme-linked immunosorbent assay (ELISA) is a specific, rapid and quantitative method, which since its introduction has been used in the identification and detection of a range of plant viral and fungal diseases. At CSL, a range of specific monoclonal antibodies (MAbs) have been raised to either the Fusarium genus or individual species: namely Fusarium avenaceum, Fusarium culmorum, Fusarium poae and Microdochium nivale (formerly Fusarium nivale) [1]. MAbs were produced with the aim of reducing the time taken and possible error involved in the identification of these species both in agar culture and directly from infected plant material. This paper concentrates on work carried out to determine the optimum age of cultures and the influence of different methods used for the extraction of antigen from infected plant material.

Materials and methods
Initially MAbs were tested on antigen produced from agar plate cultures. Potato dextrose agar plates were inoculated with the isolate from which the MAb was raised. Cultures were grown for either four or ten days at 15oC for M. nivale and 25oC for the other Fusarium species. The antigen was removed from the plates by surface washing. All ELISAs were carried out according to the protocol described in [1]. Soaking and crushing of grain and stem material were examined as possible methods of antigen extraction. The effect of each method on antigen detection was assessed by adding a known concentration of antigen to each extract prior to well coating. To identify the optimum stage of development for detection in plant material MAbs were tested using infected grain obtained from field grown plants [2]. Grain with high levels of infection was incubated in moist chambers for periods between 0 and 7 days to determine whether pre-treatment would improve antigen detection.

Results and conclusions
The successful detection of antigen from agar plate washings depended on the age of the culture. Antigen was not detected after four days incubation, however, after 10 days incubation antigen was detected at levels equivalent to the positive control. This would seem to indicate that the antigen was either present on older mycelium or produced possibly as a metabolite later in the growth of the colony. The immunological detection of Fusarium antigen from agar cultures both simplified and reduced the time required for their identification compared to conventional methods. Initial experiments using the MAbs to detect antigen directly from infected plant material were unsuccessful. The presence of crushed grain or stem material in the antigen preparation completely inhibited antigen detection. Soaking plant material reduced inhibition in comparison to crushing, with the length of soaking affecting the amount by which inhibition was reduced. Experiments using naturally infected plant material showed that the state in which antigen was present also affected detection. The MAb only detected antigen from grain which had been previously incubated in a moist chamber even though microscopic examination of the untreated grain indicated the presence of high levels of macroconidia and desiccated mycelia. The optimum incubation period was between 5 and 6 days. The MAb also detected antigen from actively growing lesions present on diseased stem-base and root material. Further refinement of the protocol, possibly by the use of more complex ELISA, is being undertaken to minimise inhibition and produce a more sensitive assay.

References
1. Banks JN, Rizvi RH, Barker I, Turner JA, Rahman S, Northway BJ, 1996. Food and Agricultural Immunology 8, 249-268.
2. Jennings P, Turner JA, 1996. Proceedings Brighton Crop Protection Conference: Pests and Diseases, pp.233-238.