Characterization of aPythium ultimum-Specific Antigen and Factors That Affect Its Detection Using a Monoclonal Antibody

Abstract

A Pythium ultimum-specific monoclonal antibody (MAb E5) recognized a 46-kDa protein band from P. ultimum in Western blots, but did not react with any protein from five other Pythium spp. Loss of MAb E5 reactivity to the antigen following treatment of the antigen with trifluoromethane sulfonic acid, periodate, or laminarinase indicated that the antigen was a glycoprotein with a carbohydrate epitope containing β-1,3 linkages. The antigen was thermostable. MAb E5 reactivity to mycelia and specific structures was affected by fungal age. The E5 antigen content in P. ultimum mycelia cultured in Czapek-Dox broth increased during the first 3 days, but subsequently declined despite continuing fungal growth. The antigen was detected in all young P. ultimum structures by immunofluorescence, but fluorescence decreased or disappeared as the structures matured or became dormant. Fluorescence reappeared upon transferring aged structures to fresh nutrient solution. Reactivity of MAb E5 to the fungus also was influenced by culture nutrients. P. ultimum cultured in media containing different seed exudates or different sugars varied in E5 antigen content. P. ultimum could not be detected in infected sugar beet and common bean hypocotyls using enzyme-linked immunosorbent assay (ELISA). The fungus was detected in infected sugar beet hypocotyls by Western blot, but not in infected bean tissue.