Abstract
The legume tree Pongamia pinnata (also called Millettia pinnata) is a non-food crop that can grow on marginal land not destined for the cultivation of food crops. It is an important candidate for the production of biofuel (bio-oil, biodiesel and aviation biofuel) from its oil-rich seeds. An important trait is its ability to grow in marginal, nitrogen-limited soils. This growth ability is attributed to the nitrogen-fixation activity of root nodules. Cutting both cotyledons in halves had little effect on shoot dry weight, nodule number and weight per plant at 8 weeks. These parameters were reduced when both cotyledons were removed; plants were visibly yellow. Surprisingly when no nutrients were supplied and with both cotyledons intact, plants grew as well as those with nutrients. To isolate rhizobial inoculants from soil, samples were collected from Meandu Mine, a coal-mining site near Kingaroy, Queensland (Australia), where pongamia is currently grown to rehabilitate the soil. Pongamia nodules were also collected from Mt. Coot-tha Botanic Gardens (Brisbane), where pongamia was introduced decades ago. Established ‘Baiting technique’ and ‘One-drop-one-nodule technique’ were applied to isolate potential rhizobia from the soil samples and nodules. All isolates were screened and characterised for symbiotic effectiveness and nitrogenase activity. A total of 21 putative rhizobial samples were isolated – five from soil samples and 16 from pongamia nodules. Out of these iso lates, seven were slow-growers while 14 were fast-growers. These isolates were coded as PR-UQ. Among the isolates, the fast-growers PR-UQ-03 and PR-UQ-05 were considered as the superior strains compared to the slow-growers PR-UQ-01 and PR-UQ-04, which were more effective than the rest of the isolates. The new isolates enhanced nodule number, shoot length and total plant dry weight and resulted in better plant growth than the available Bradyrhizobium japonicum strains CB1809, USDA110 and CB564. Acetylene reduction assay (short term) and Nitrogen Difference Analysis (long term) further confirmed that pongamia fixed more nitrogen upon inoculation with these rhizobia. Based on 16S rDNA sequence analysis, PR-UQ-01 and PR-UQ-04 are closely related to Bradyrhizobium elkanii and Bradyrhizobium pachyrizi, while PR-UQ-03 and PR-UQ-05 are both related to Rhizobium mesoamericanum. Cotyledons supply the seedling with nutrients for a considerable period of early seedling growth. The newly isolated, fast-growing rhizobia PR-UQ-03 and PR-UQ-05 and the slow-growing PR-UQ-01 and PR-UQ-04 are related to Rhizobium and Bradyrhizobium species and aid in the nodulation, nitrogen fixation and seedling/sapling growth of Pongamia pinnata.
Highlights
Pongamia pinnata, a fast growing, outcrossing, papillionaceous tree legume with high oil content seeds found in northern Australia, Papua New Guinea, Indonesia and India, is a potential feedstock for biofuel production (Biswas et al, 2013; Gresshoff et al, 2015; Scott et al, 2008)
This study aimed to identify effective rhizobia that can nodulate and fix nitrogen with pongamia in Queensland, Australia
To determine the role of cotyledons in nodulation and avoid the carry-over of nutrients from cotyledons, 40 pongamia seedlings growing in sterile vermiculite at 20 days old were divided into five treatments: (1) inoculated with CB1809 but no nutrient solution added; (2) uninoculated and no nutrient solution added; (3) inoculated with CB1809 with nutrient solution (B&D N-free solution); (4) uninoculated but with nutrient solution; and, (5) uninoculated but with nitrogen (2 mM ammonium nitrate solution) and nutrient solution
Summary
A fast growing, outcrossing, papillionaceous tree legume with high oil content seeds found in northern Australia, Papua New Guinea, Indonesia and India, is a potential feedstock for biofuel production (Biswas et al, 2013; Gresshoff et al, 2015; Scott et al, 2008). It is salinity and drought tolerant (especially when established beyond the seedling stage), and grows well in low fertility soils (Kazakoff et al, 2011). This legume tree produces seeds, which contain about 40% (w/w) oil, predominantly 50% of which is the mono-unsaturated oleic acid (C18:1) that can be used for biodiesel production (after transesterification with methanol or ethanol; Kazakoff et al 2011). Pongamia oil is made up predominantly of triglycerides, which can be converted by transesterification to biodiesel (fatty acid methyl esters; FAME) or by hydrogenation to aviation A1 jet fuel (Kazakoff et al, 2011; Klein-Marcuschamer et al, 2013)
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