Determination of toxin distribution in Alternaria leaf spot diseased tissue by radioimmunoassay

Abstract

A radioimmunoassay for the phytotoxin brefeldin A has been developed employing [7-³H] brefeldin A and an antiserum raised against 7-dehydrobrefeldin A conjugated to bovine serum albumine. The antisera allowed the determination of as little as 1 pmol of toxin and had a similar affinity for both 7-epi-brefeldin A and 7-dehydrobrefeldin A. Several other compounds, including some with structures similar to brefeldin A and certain toxins from other Alternaria species, were bound by the antiserum but at least 6000-fold less strongly than brefeldin A.

The radioimmunoassay was used to determine the amounts of brefeldin A in leaf tissues adjacent to and distant from the inoculation site in safflower leaves inoculated with the pathogen Alternaria carthami or with the non-pathogens Ascochyta imperfecta and Eupenicillium brefeldianum. High concentrations of brefeldin A, up to approximately 3 mM, accumulated in leaf tissues over a period of 17 days after inoculation with A. carthami. The other two fungi, although able to produce large quantities of brefeldin A in stationary culture, did not accumulate it in inoculated leaves and the small amount of toxin present with the inoculum disappeared with time. Alternaria carthami, on the other hand, failed to accumulate the toxin when inoculated onto leaves of Zinnia elegans, Helianthus annus, Calendula officinalis or Lactuca sativa.

These results support our earlier hypothesis that efficient production of brefeldin A is a factor in the mechanism of infection of safflower tissue by the pathogen A. carthami.