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Integrating field and physiological studies to understand acid tolerance in Rhizobium leguminosarum bv. trifolii

Watkin, Elizabeth, L.J. (1999) Integrating field and physiological studies to understand acid tolerance in Rhizobium leguminosarum bv. trifolii. PhD thesis, Murdoch University.

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The work in this thesis aims to evaluate the physiological differences between acid-soil tolerant and acid-soil sensitive strains of R. trifolii. The poor correlation between laboratory screening for acid tolerance in root nodule bacteria and field performance necessitates the use of time consuming field trials for the selection of acid tolerant strains. By gaining an increased understanding of the physiological basis of acid tolerance in root nodule bacteria, reliable laboratory based selection criteria for acid-soil tolerance may be established.

A field experiment was performed at Agriculture Western Australia Drylands Research Institute, Merredin in an acid soil (pH 4.2, 0.01 M CaCl2) to determine the ability of six strains to survive in, and colonise this site. Strains NA3039 and WSM409 survived and colonised significantly better than strain NA3001 and TA1. The study of the effect of acidity on the growth and survival, in laboratory media, of the six strains used in the field experiment showed that the performance of these strains in an acid soil was contrary to their ability to grow in an acidic medium with strain NA3001 able to grow at a more acidic pH than the other five strains and the growth NA3039 appearing to be the most affected by an acidic medium.

The interaction of acidity and calcium on the growth and survival of three of the strains was studied in the presence of high (300 µM) and low (20 (µM) concentrations of phosphate. A region of "acid-stress" somewhere below pH 5.0 was observed where growth rate slowed rapidly over 0.2-0.3 of a pH unit. The presence of 300 µM phosphate did not affect the critical pH for growth or growth rate within the "acidstress" zone but did reduce the mean generation time of all strains studied at pH above the "acid-stress" zone. At pH 7.0 increasing calcium from 300 µM to 3000 µM had little effect on growth rate, but high calcium increased growth rate within the "acid-stress" zone and enabled growth at a lower pH than that observed with the low calcium concentrations.

From the field experiment an acid-soil tolerant and an acid-soil sensitive strain were selected for detailed physiological examination. Phosphate limited chemostats were used to investigated the effect of protons on these two strains. Electron microscopy showed that acidity affected the morphology of the acid-soil tolerant strain, whereas acidity did not change the morphology of the acid-soil sensitive strain.

Both strains had greatly reduced motility at pH 4.8 as compared to that at pH 7.0. Cells transferred from pH 7.0 to pH 4.8 showed an immediate reduction in the number of motile cells, with the acid-soil tolerant strain being less affected than the acid-soil sensitive strain. When transferred from pH 4.8 to pH 7.0 there was an immediate increase in motility with levels returning to those observed at pH 7.0 within 3-4 generations.

Low pH resulted in both strains having significantly higher total phosphate and lower total potassium than at neutral pH. Both calcium and magnesium were present at much lower levels at low pH for both strains.

The acid-soil tolerant strain induces an adaptive acid tolerance response; cells grown at pH 4.8 had greater acid tolerance than cells grown at pH 7.0. The acid-sensitive strain showed an acid sensitive response.

The conclusion of this work is that acid-soil tolerant strain may be able to recognise increasing acidity and respond by invoking systems to cope with this stress. There is no evidence that the acid-soil sensitive strain has this ability.

Item Type: Thesis (PhD)
Murdoch Affiliation: Division of Science
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): O'Hara, Graham, Howieson, John and Glenn, Andrew
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