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

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.
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