|Plaisance, K - UNIVERSITY OF MINNESOTA|
Submitted to: Weed Science Society of America Meeting Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: February 7, 2000
Publication Date: N/A
Technical Abstract: Previous research demonstrated the potential of Pseudomonas syringae pv. tagetis (Pst) as a biological agent for controlling Canada thistle in soybean. The apical chlorosis caused by Pst is due to the production of tagetitoxin, a RNA polymerase III inhibitor that blocks chloroplast biogenesis. A better understanding of factors regulating tagetitoxin production in Canada thistle is needed to increase efficacy of Pst as a biocontrol agent. We have been developing a protocol to purify tagetitoxin with the goal of generating an antibody to be used to monitor toxin production in planta. When grown in modified Wolley's medium to a population of approximately 10**10 cfu/ml, Pst strain EBO 34 produces tagetitoxin in culture. The culture supernatant was applied to an anion- exchange (QAE Sephadex A-50) column and eluted with increasing ionic strength (50 to 400 mM ethanolamine). Fractions containing tagetitoxin were eidentified by using either a sunflower bioassay or an in vitro RNA polymerase inhibition assay. Fractions containing tagetitoxin were combined and applied to an adsorption column (LH20 Sephadex) fitted with a refractive index detector. When eluted with methanol: 50 mM ammonium hydroxide (50:50, v/v), a single peak of tagetitoxin was detected. Based on the in vitro RNA polymerase inhibition assay, the purification of tagetitoxin was greater than 1000-fold.