Altered phenolic activity in Pinus elliottii cell cultures challenged with the fungus, Ceratocystis minor

Abstract

A plant cell culture system was developed to study biochemical responses of Pinus elliottii Engelm. (slash pine) cells to Ceratocystis minor (Hedgc.) Hunt, a fungal parasite of southern pines. Early experiments showed that the living fungus and its culture filtrate caused necrosis in cultured pine cells. Also an increase in syringaidazine oxidoreductase activity was present after prolonged interaction between pine cell cultures and the fungus. In suspension cultures, this activity was identified as being due to peroxidase, not laccase. A variety of possible defense compounds were explored for potential study, including monoterpenes, callose, soluble phenolics, and lignin. Total phenolics and lignin were chosen for more extensive study because they were altered upon fungal challenge to the cells in culture and because analyses were more accessible. Slash pine calli challenged with live fungus showed a significant increase in lignin staining over non-challenged calli, with the degree of increased lignin staining decreasing with callus age. In stationary pine cell suspension culture, the fungus was found to cause increased lignin staining, decreased alcohol-soluble total phenols, and altered extracellular peroxidase activity. A characteristic feature of the fungal-treated pine cell cultures was an accumulation of extracellular material (ECM) that stained for peroxidase activity as well as lignin. In the stationary pine cell culture treated with fungus, there was a strong correlation between increased lignin-stained material and a drop in total phenols in both cell extracts and culture filtrates. As early as 3 h post-treatment, a drop in total phenols could be detected in culture filtrates of fungal-treated pine cell suspension cultures. ECM was apparent in culture filtrates as early as 3 h, but was more prevalent at 6 h post-treatment. Increased lignin-stained material and a drop in total phenols could also be induced by treatment with an autoclaved C. minor elicitor preparation. Attempts were made to determine if seedlings responded to C minor in a similar manner as the cell cultures. Inoculated seedling stems became necrotic in response to the fungus, while wound-only stems appeared to heal completely; however, both woundonly and wound-inoculated stems showed lignin staining at the wound site.