Date of Award:

5-1-1998

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Committee Chair(s)

Bradley R. Kropp

Committee

Bradley R. Kropp

Committee

Anne J. Anderson

Committee

Jeanette Norton

Abstract

Phenazines, the water-soluble pigments produced naturally by bacteria with distinct antimicrobial properties, have been implicated in the biological control of several plant diseases. Pseudomonas chlororaphis strain O6, obtained from the rhizosphere of wheat, is known to produce at least three different phenazines, which include phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. A field trial conducted in the fall of 1994 to test the biocontrol properties of O6 indicated that this strain inhibited wheat seed germination, which led me to hypothesize that phenazines cause phytotoxicity. Mutants of O6 created by Tn5 lux insertion (Phz- mutants) that did not produce the pigments characteristic of phenazine, not only lost their ability to inhibit the wheat pathogen Fusarium culmorum, but also their phytotoxic properties. In order to test their effect on seed germination, the wild-type strain and the different Phz- mutants were inoculated onto wheat seed after being grown in different media. The wild-type still caused phytotoxicity symptoms even when grown in a medium not conducive for phenazine production. This indicated that nutrients exuded during wheat seed imbibition and germination were sufficient to support phenazine biosynthesis. An inoculum dosage effect was also seen in treatments involving phenazine production. Decreasing levels of phenazines brought about by the dilution of inoculum corresponded to increased seed germination. The mutants of O6 were not affected in their ability to produce hydrogen cyanide. Hence it was concluded that the phytotoxicity symptoms observed during the course of this study were not due to HCN production. Southern analysis of the Phz- mutants revealed that all of them had a single insertion of the Tn5 luxAB. Complementation of one of the Phz- mutants (L21) with cloned sequences from a library of the O6 wild-type DNA restored pigmentation associated with phenazine production, fungal inhibition in vitro, and inhibition of wheat seed germination to wild-type levels.

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