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Identification and characterization of root nodule bacteria from South-Western Australia soils able to nodulate Acacia saligna

Marsudi, Nada D.S. (1997) Identification and characterization of root nodule bacteria from South-Western Australia soils able to nodulate Acacia saligna. Masters by Research thesis, Murdoch University.

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Abstract

One hundred and thirty root nodule bacterial strains were isolated from nodules of Acacia saligna grown in ten different soils from nine locations in South- Western Australia. These isolates were confirmed as root nodule bacteria by reinoculation onto their host plant (A. saligna). Based on their morphological characteristics and growth rates on yeast mannitol agar (YMA), thirty nine strains were classified as fast-growing rhizobia and ninety four as slow-growing bradyrhizobia. The fast-growing strains were found in all soils except one from Narrikup which had the lowest pH (4.62).

Selected strains (20 fast-growing and 20 slow-growing) were characterized for their ability to grow:
•at acid pH (5 ± 0.2), or at alkaline pH (8.8 ±0.1)
•at 37°C
•on mannitol, fructose, arabinose, lactose or sucrose as sole carbon sources
•in the presence of 1 -3% (w/v) NaCl

The slow-growing bradyrhizobia were more acid-tolerant than the fastgrowing rhizobia, while the fast-growing rhizobia were more alkali tolerant than the slow-growing bradyrhizobia. Most fast-growing rhizobia grew at 37°C, whereas only two slow-growing bradyrhizobia could grow at this temperature. Both fast- and slowgrowing root nodule bacteria grew on mannitol, fructose and arabinose as a sole carbon source. All of the fast-growing rhizobia utilized the disaccharides (lactose and sucrose), whereas the slow-growing bradyrhizobia did not. All the slow-growing bradyrhizobia were sensitive to salt and did not grew even at 1% (w/v) NaCl. All of the fast-growing rhizobia grew at 1% NaCl and some at 3% NaCl.

In order to investigate the diversity within a segment of the 16S rRNA genes of these root nodule bacterial strains, 10 fast-growing rhizobial strains and 10 slowgrowing bradyrhizobia were selected, with Sinorhizobium meliloti WSM 419 as a control strain. A 260-264 bp segment of DNA from each strain (corresponding to positions 44 to 337 in the Escherichia coli 16 S rRNA gene) was amplified using the polymerase chain reaction (PCR) method and the amplified DNA sequenced. The 16S rRNA (DNA) sequences were then compared and aligned to those known for reference root nodule bacterial strains from the GenBank Data Library leading to a phylogenetic unrooted tree for the strains from A. saligna.

Analysis of the 16S rRNA gene sequences confirmed that the root nodule bacterial strains able to nodulate A. saligna can be separated into fast- and slow-growing strains.

The fast-growing rhizobia can be divided into two groups. The first group consists of six strains which showed 96.5-100% similarity over 260 bp to Rhizobium leguminosarum biovar phaseoli U29388. The second group consists of four strains which showed 97.7-100% similarity over 260 bp to Rhizobium tropici U38469. The slow-growing bradyrhizobia could also be grouped together. Six strains showed 97.3- 99.2% similarity over 264 bp to Bradyrhizobium japonicum Z35330. Three strains showed 99.2-100% similarity over 264 bp to Bradyrhizobium sp. (Lupinus) X87273 and strain YL68 showed 100% similarity over 262 bp to Bradyrhizobium sp. (Lupinus) U69636. S. meliloti WSM419 showed 100% similarity over 260 bp to Sinorhizobium sp. Z78204.

Item Type: Thesis (Masters by Research)
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: repository@murdoch.edu.au. Thank you.
Supervisor(s): Dilworth, Michael and Glenn, Andrew
URI: http://researchrepository.murdoch.edu.au/id/eprint/51836
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