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Rapid evolution of diversity in the root nodule bacteria Biserrula plecinus L

Nandasena, Kemanthi Gayathri (2004) Rapid evolution of diversity in the root nodule bacteria Biserrula plecinus L. PhD thesis, Murdoch University.

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Biserrula pelecinus L. has been introduced to Australia from the Mediterranean region, in the last decade due to many attractive agronomic features. This deep rooted, hard seeded, acid tolerant and insect resistant legume species provides high quality food for cattle and sheep, and grows well under the harsh edaphic and environmental conditions of Australia. In 1994, B. pelecinus was introduced to a site in Northam, Western Australia where there were no native rhizobia capable of nodulating this legume. The introduced plants were inoculated with a single inoculant strain of Mesorhizobium sp., WSM1271. This study investigated whether a diversity of rhizobia emerged over time. A second objective was to investigate the possible mechanisms involved in the diversification of rhizobia able to nodulate B. pelecinus.

Eighty eight isolates of rhizobia were obtained from nodules on B. pelecinus growing at the Northam site in August 2000, six years after introduction. These plants were self-regenerating offspring from the original seeds sown. Molecular fingerprinting PCR with RPO1 and ERIC primers revealed that seven strains (novel isolates) had banding patterns distinct from WSM1271 while 81 strains had similar banding patterns to WSM1271. A 1400 bp internal fragment of the 16S rRNA gene was amplified and sequenced for four of the novel isolates (N17, N18, N45 and N87) and WSM1271. The phylogenetic tree developed using these sequences clustered the novel isolates in Mesorhizobium. There were >6 nucleotide mismatches between three of the novel isolates (N17, N18, N87) and WSM1271 while there were 23 nucleotide mismatches between N45 and WSM1271.

When B. pelecinus cv. Casbah was inoculated with the novel isolates, five (N17, N18, N39, N46 and N87) yielded <40% of the shoot dry weight of the plants inoculated with the original inoculant (WSM1271). Novel isolates N15 and N45 were completely ineffective on B. pelecinus cv. Casbah.

Physiological experiments to test the ability of the novel isolates and WSM1271 to grow on 14 different carbon sources (N acetyl glucosamine, arabinose, arbutine, dulcitol, beta-gentiobiose, lactose, maltose, melibiose, D-raffinose, saccharose, L-sorbose, D-tagatose, trehalose and D-turanose) as the sole source of carbon, intrinsic resistance to eight different antibiotics (ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, spectinomycin, streptomycin and tetracycline) and pH tolerance (pH 4.5, 5.0, 7.0, 9.0) revealed that the novel isolates had significantly different carbon source utilization patterns to WSM1271. However, pH tolerance and intrinsic resistance to antibiotics were similar between the novel isolates and WSM1271 except for streptomycin (100 mcg/ml). Novel isolates N17, N18, N46 and N87 were susceptible for this antibiotic while the other novel isolates and WSM1271 were resistant.

Host range experiments were performed for the novel isolates N17, N18, N45, N87, WSM1271 and two other root nodule bacteria (RNB) previously isolated from B. pelecinus growing in the Mediterranean region (WSM1284 and WSM1497) for twenty one legumes (Amorpha fruticosa, Astragalus adsurgens, Astragalus membranaceus, Astragalus sinicus, Biserrula pelecinus cv Casbah, Dorycnium hirsutum, Dorycnium rectum, Glycyrrhiza uralensis, Hedysarum spinosissimum, Leucaena leucocephala, Lotus corniculatus, Lotus edulis, Lotus glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus parviflorus, Lotus pedunculatus, Lotus peregrinus, Lotus subbiflorus, Macroptilium atropurpureum, and Ornithopus sativus). Only isolate N17 have the same host range as WSM1271 in that they both nodulated B. pelecinus and A. membranaceus, while the other three novel isolates, WSM1284 and WSM1497 had a broader host range than WSM1271. Three isolates N18, N45 and N87 formed small white nodules on M. atropurpureum, in addition to nodulating the above hosts. Isolates N18 and N45 also nodulated A. adsurgens while N45 was the only isolate to nodulate L. edulis. Isolate N87 was the only isolate to nodulate A. fruticosa. WSM1497 nodulated A. adsurgens, A. membranaceus, B. pelecinus and L. corniculatus while WSM1284 was a promiscuous strain that nodulated 16 host species out of the 21 tested. A 710 bp internal region of nifH, a 567 bp internal region of nodA and a 1044 bp internal region of intS were sequenced for N17, N18, N45, N87 and WSM1271. The sequence comparison showed that the sequences of the above three genes of the four novel isolates were identical to that of WSM1271.

Eckhardt gel electrophoresis revealed that WSM1271, three other RNB isolates from B. pelecinus from the Mediterranean region and isolate N18 each have a plasmid of approximately 500 kb while N17, N45 and N87 are plasmid free. Probing of the plasmid DNA from the Eckhardt gel with nifH and nodA probes indicated that these two genes were not located on the plasmid.

Furthermore, the results of this study demonstrated that 92% of the nodules on B. pelecinus growing in the Northam site six years after the introduction of this plant were occupied by the inoculant strain and the N2 fixation efficiency of the progeny strains of WSM1271 remain similar to the mother culture. This study also showed that the carbon source utilization pattern, intrinsic antibiotic resistance and pH range of the progeny strains of WSM1271 remain relatively similar, except for few variations in carbon source utilization patterns.

This thesis clearly demonstrated that phenotypicaly, genetically and phylogenetically diverse strains capable nodulating B. pelecinus evolved through symbiotic gene transfer from the inoculant strain to other soil bacteria within six years. The presence of intS, and the evidence of gene transfer between these Mesorhizobium strains indicates that transfer of symbiotic genes may have occurred via a symbiosis island present in WSM1271.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): O'Hara, Graham
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