IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

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17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Session Details: Thursday 1 December 2011 Concurrent Session 17 – Molecular Characterization of N-fixing organisms 1100 - 1230 Authors: Melissa K. Corbett, Lesley A. Mutch and Elizabeth L.J. Watkin School of Biomedical Sciences, Curtin University, Kent Street, 6845, Western Australia Presentation Title: Identification and evolution of NIF genes in Leptospirillum species – an acidophilic chemolithoautotroph Presentation Time: 1140 – 1200 Leptospirillum are gram negative, chemolithoautotrophic extremophiles found in environments where sulfide and iron bearing minerals are exposed to the air. Their natural ability to oxidize ferrous iron for energy, leaving behind other liberated minerals has been industrialized in the form of biomining. Biomining operations rely on this microbial aided extraction of metals from solid minerals, but mineral recovery rates can be affected if nutrient levels fluctuate and gradients form. The addition of ammonium based chemicals to meet Leptospirillum growth needs can be expensive, and equal distribution is not guaranteed. Genomic research has uncovered that two of the three documented species, L. ferrooxidans and L. ferrodiazotrophum encode genes for nitrogen fixation. To ascertain whether the third species, L. ferriphilum, also has the potential for diazotrophy, degenerate nifH primers were tested. A single PCR product of approximately ~900bp was amplified and subsequent sequence analysis revealed significant identity (84%) to the nifH genes in the other Leptospirillum species. Phylogenetic analysis of the single nifH genes placed all Leptospirillum species in the conventional Mo-containing nifH cluster I, closely related to the nifH genes of �-proteobacterium, Acidithiobacillus, a known nitrogen fixing biomining microbe. Concatenated super-gene alignment of the nifH gene with 16SrRNA, gyr B, nifD and nifK provided a greater resolution of evolution amongst the closely related strains. The use of maximum likelihood and maximum parsimony phylogenetic trees further elucidated the evolutionary relationships between the three Leptospirillum species. Discovery of these genes in all identified Leptospirillum species aids in cementing the status of the Leptospirillum genus as free living diazotrophs whose presence in biomining operations can help sustain mineral leaching rates when soluble nitrogen levels are low, without the incursion of additional operational costs. 106 2011
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Session Details: Thursday 1 December 2011 Concurrent Session 17 – Molecular Characterization of N-fixing organisms 1100 - 1230 Authors: Ni Luh Arpiwi1 , Elizabeth LJ Watkin2 , Guijun Yan1 , Elizabeth L Barbour, 1, 3 and Julie A Plummer 1 1 School of Plant Biology, University of Western Australia, 35 Stirling Hwy Crawley WA 6009 2 School of Biomedical Science, Curtin University, Bentley WA 6102 3 Forest Products Commission of Western Australia, 117 Great Eastern Highway Rivervale, WA 6103, Australia. Presentation Title: Physiological and molecular characterization of the root nodule bacteria nodulating Millettia pinnata, a biodiesel tree Presentation Time: 1200 – 1220 Milletia pinnata is a leguminous tropical tree that produces seed oil suitable for biodiesel. By optimizing the symbiotic relationship with nitrogen fixing bacteria reduced fertilizer inputs would be required, which would be advantageous for this second generation biofuel crop, especially for production on marginal land. This study aimed to isolate root nodule bacteria from soil around Millettia trees, confirm their symbiotic potential, examine their physiological characteristics and to identify elite strains. Soil samples were collected from the base of Millettia pinnata grown in plantations in northern, tropical Western Australia. Seeds were planted in pots layered with 3 cm of sterile coarse sand then 3 cm soil, 2 cm sterile coarse sand and 1 cm sterile plastic beads on the top. Seeds were placed in the upper coarse sand layer and grown for 18 – 20 weeks. Root nodules were harvested and crushed, and the contents streaked onto a half-lupin agar plates and incubated at 29 o C. Forty pure isolates were obtained which produced single colonies in 5-9 days. Optimum growth conditions for these isolates were pH 7 – 9 and 29 – 37 o C. Growth of all isolates was reduced at 1 – 2% NaCl and no isolates grew at 3% NaCl. Most isolates had optimal growth on mannitol, arabinose or glutamate as a single carbon source, only a few grew on sucrose and none grew on lactose. Cluster analysis of isolates based on physiological characteristics indicated phenotypic diversity. Isolates were re-authenticated and the effectiveness of the symbiotic association assessed based on shoot dry weight compared to that of the nitrogen-fed control plants. Genetic diversity of isolates was analyzed and confirmed using the sequence of the 16S RNA gene. The outcomes of this research will promote the growth of Millettia plantations in nutrient poor soils which alleviates competition on arable lands for food production. 107 2011
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IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

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