Masters Theses

Date of Award

5-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Entomology and Plant Pathology

Major Professor

Bonnie H. Ownley

Committee Members

Neil Quigley, Mark Windham, Craig Canaday

Abstract

Take-all, caused by Gaeumannomyces graminis var. Tritici (Ggt), is a root and crown disease of wheat that can have devastating effects on crops in temperate grain-producing areas throughout the world. Many antibiotic-producing strings of the bacterial genus Pseudomonas have been shown to effectively suppress Ggt in vitro, and to control take-all in greenhouse and field studies. When applied as seed treatments, Pseudomonas fluorescens strain Q2-87 and P. fluorescens stain 2-79 control take-all, primarily by antibiosis. Strain Q2-87 produces the broad-spectrum antibiotic 2,4- diacetylphloroglucinol (PhI), and strain 2-79 produces phenazine-1-carboxylic acid (Phz). Before strains Q2-87 or 2-79, or similar biocontrol seed treatments can be made commercially available, their effects on nontarget organisms such as beneficial soil bacteria, must be characterized.

For studies on the nontarget effects of P. fluorescens strain Q2-87 to be most useful, it was required that a high level of microbial activity take place among the nontarget wheat rhizosphere bacteria in the growth chamber assays. To encourage high microbial activity, the nontarget experiments were to be conducted at a temperature range of 20 to 25 C. It was also necessary that the introduced strain, Q2-87, exhibit high antibiosis activity at the temperature range selected for the nontarget study. The effect of temperature (5, 10, 15, 20, 25, 30 C) on antibiosis activity of P. fluorescens strain Q2-87 (PhI+, HCN+) as evidenced by in vitro inhibition of Ggt was studied to determine its activity at the nontarget assay temperature range of 20 to 25 C. P. fluorescens Q2-87::Tn5 (PhI+, HCN+), a transposon mutant derived from Q2-87, which lacks the ability to produce PhI was included in this study for comparison. Production of HCN by both strains were observed also at the temperature range of 5 to 30 C.

In the absence of PhI+ or PhI- bacteria, Ggt grew at all termpatures (5, 10, 15, 20, 25, 30 C) tested. Growth was greatest at 20 and 25 C. P. fluorescens strain Q2-87 inhibited growth of Ggt in vitro at all temperatures tested, however, inhibition was greatest at 20 and 25 C. A low level of inhibition was exhibited by the PhI- strain, Q2-87::Tn5, at all temperatures tested, however, there were no differences in the degree of inhibition across the temperature range tested. Inhibition of mycelial growth of Ggt by the PhI+ strain was significantly greater than the PhI- strain at 20 and 25 C. Based on a qualitative comparison, production of HCN by Q2-87 and Q2-87:;Tn5 increased with temperature, and was greatest at 25 and 30 C. This study affirmed that nontarget Conetainer assays involving strains Q2-87 and Q2-87::Tn5 could be conducted at 20-25 C.

In two experiments, the nontarget effects of introduced, antibiotic-producing fluorescent pseudomonad biological control bacteria on selected rhizosphere bacteria were investigated. The primary objectives of these experiments were 1) to monitor populations of selected, culturable wheat rhizosphere bacteria, and to determine changes in those populations after the addition of antibiotic-producing biocontrol fluorescent pseudomonads, 2) to determine the ability of biocontrol strains Q2-87 and 2-79 and their respective antibiotic-deficient mutants to persist in soil, and 3) to determine the effect of cropping sequence and tillage on objectives 1 and 2. The tillage regimes were simulated tillage and no-till. The cropping sequences were wheat-fallow and wheat-soybean. The bacterial seed treatments were untreated wheat seed, or seed treated with P. fluorescens strains Q2-87.8283 (PhI+), Q2-87::Tn5.2793 (PhI-), 2-79 (Phz+), or 2-79.2A40 (Phz-).

All experiments were performed in a growth chamber with soil placed in Conetainers. Experiments with each tillage regime were conducted separately. All cones were cropped to three cycles of wheat, followed by a wheat-soybean cycle or a wheat-fallow cycle. Then the treated seeds were planted. Bacterial populations in the wheat rhizosphere were assessed at the 3-4 leaf stage using dilution plating methods and eight types of bacteriological media. Following the first sampling (cropping cycle 1), untreated wheat seed was planted in cones that were not sampled after the first cycle. With wheat at the 3-4 leaf stage, plants were sampled for cropping cycle 2.

After the first cropping cycle of both tillage regimes, population densities of actinomycetes, Arthrobacter, Azotobacter, Bacillus, free-living nitrogen-transformation bacteria, and total aerobic bacterial populations were not reduced by seed bacterization with PhI+, PhI-, Phz+, Phz- strains. Total fluorescent pseudomonad densities were greater on roots from seed treated with PhI+, PhI-, Phz+, Phz- strains than the check-treated roots. The introduced pseudomonads accounted for a high proportion of the total pseudomonads recovered after the first cycle.

Production of PhI did not confer a colonization advantage, as the PhI-deficient mutant colonized the roots equally well as the PhI+ parent strain. In contrast, in the no-till study, population numbers of Phz+ bacteria were greater than Phz- bacteria after the first cropping cycle.

After a second cycle with either a soybean planting or a fallowing, and a planting of non-bacterized wheat, indigenous pseudomonads recovered to populations densities previously observed, and introduced strains declined to log 1 or less.

In many instances, populations of selected nontarget bacteria were affected by cropping sequence. In general, populations were lower in soil previously fallowed than in soil previously planted to soybean. The previous cropping sequence (wheat-fallow or wheat-soybean) did not affect densities of the introduced Q2-87.8283 or Q2-87::Tn5.2793 strains recovered from wheat roots, but a wheat-soybean sequence did tend to favor 2-79 or 2-79.2A40 strains.

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