Investigation of the symbiotic associations of Acacia ligulata Benth. and Acacia tetragonophylla F.Muell: The potential for use in the rehabilitation of excavated sites at Shark Bay Salt Pty. Ltd.

Yvette Hill

The integration of plant available nitrogen (N) into the nutrient cycles of dryland ecosystems is integral to the establishment and persistence of the flora in these regions. Much of this available N is due to the conversion of atmospheric dinitrogen (N2) by legumes and their bacterial microsymbionts, root nodule bacteria (RNB). There are numerous environmental constraints in dryland areas that impede the growth and interactions of both symbiotic partners. At Shark Bay Salt Pty. Ltd., a solar salt facility in Western Australia, the associations between provenant RNB and the key over-story species Acacia ligulata Benth. and Acacia tetragonophylla F.Muell. were investigated in situ and in glasshouse conditions. This was done to determine whether the selection of provenant RNB that effectively fix nitrogen, and their inoculation onto these two species, could improve plant establishment at degraded pit sites within the Shark Bay Salt lease area (SBSLA). The effect that mining processes has had on the biological, chemical and physical characteristics of the remaining substrate of selected borrow pit soils was evaluated. The removal of the soil, subsoil and regolith had altered the chemical characteristics of these sites in comparison to adjacent undisturbed areas. This activity had been deleterious to the biota, with no established floral community and reduced populations of RNB that nodulate A. ligulata Benth. and A. tetragonophylla F.Muell. in the pit areas. There was reduced organic carbon, nitrate and phosphorus concentrations in the pit soils in comparison to the adjacent undisturbed soils and at one pit site, soil salinity was at toxic levels. There were marked differences in the floristic structure and diversity between the different undisturbed sites, with A. ligulata Benth. and A. tetragonophylla F.Muell. identified at all the selected sites. The RNB in the soils was assessed in 2007 and 2008, years with contrasting annual rainfalls of 79.3 mm and 513.6 mm and it was found that the RNB population increased with the higher rainfall in all pit and undisturbed site soils, with the exception of the toxic saline pit soil where RNB were not detected. In both years, the most probable number (MPN) of RNB that nodulated A. ligulata Benth. and A. tetragonophylla F.Muell. were reduced in the pit soils compared to the adjacent undisturbed soils. Provenant isolates of RNB from the soils of SBSLA were collected and assessed for the effectiveness of these RNB isolates as well as Wattle Grow™ in promoting the growth of selected host species in glasshouse conditions for 56 day post inoculation (dpi). Many of the RNB isolated from A. ligulata Benth. and A. tetragonophylla F.Muell. readily cross-infected these two species and a number of strains also nodulated with Acacia rostellifera Benth. and Templetonia retusa (Vent.)R.Br.. There was a significant growth response of A. ligulata Benth., A. rostellifera Benth. and A. tetragonophylla F.Muell. to inoculation with a number of the RNB in comparison to uninoculated plants, with some producing foliage weights greater than 100% of the nitrogen-fed control. A. ligulata Benth. and A. rostellifera Benth. produced significantly increased growth when inoculated with Wattle Grow™ (containing Bradyrhizobium spp.). The nitrogen concentrations of A. ligulata Benth. and A. tetragonophylla F.Muell. foliage of selected treatments showed a weakly positive, non-significant relationship when correlated to the plant dry foliage weights of these treatments. While only nine RNB isolates were obtained from nodules collected from A. ligulata Benth. plants growing within the SBSLA, 78% produced a significant growth response. In contrast, only 22% of 32 A. ligulata Benth. isolates trapped from soil collected from SBSLA produced a significant growth response in comparison to the uninoculated control. This indicates a possible selection pressure and bias when trapping RNB from soils in glasshouse conditions opposed to collecting RNB directly from nodules formed on legumes at the field site. No RNB symbionts of A. ligulata Benth. and A. tetragonophylla F.Muell. have previously been described and the phenotypic characteristics, phylogenetic relationships and the genetic diversity of 25 SBSLA RNB isolates of these Acacia spp. were assessed. The RNB showed tolerance of alkaline, saline and high temperature conditions. All grew at pH 11.0 and the majority tolerated up to 750 mM NaCl. With the exception of two isolates, all grew at 37°C and five isolates were able to grow at 42°C. Based on RPO1-PCR fingerprints, there were indications of considerable genetic diversity among the RNB isolates. The 16s rDNA restriction patterns produced by Alul, Mspl and Sau3Al digestions grouped the isolates into one of six RFLP type groups. On determining the phylogeny of ten of the isolates, the 16s rDNA sequences aligned within the Ensifer, Rhizobium and Neorhizobium genera. Eight of the isolates aligned within Ensifer, six of which formed a distinct cluster. A multi-locus approach of conserved gene regions would need to be examined to more confidently assess the phylogeny of these RNB. Based on the effectiveness results, a number of RNB were selected to be re-introduced into selected pit sites in seeding and inoculation trials. Coupled with these trials, different carriers for the RNB were also evaluated to determine their efficacy in relation to the nodulation and growth response of A. ligulata Benth. and A. tetragonophylla F.Muell. in the field conditions. There was increased nodulation of A. ligulata Benth. and A. tetragonophylla F.Muell. plants that had been inoculated. The number of germinated plants and the inoculant treatment indicated no significant relationship. However, seeds inoculated with the peat treatment did generally have a greater number of plants that were growing at the assessment periods compared to the other carriers and uninoculated treatments. This itself is noteworthy as reducing seed loss is one of the major impediments to successful rehabilitation of dryland areas. The nodules on the Acacia spp. grown in the pits were occupied by RNB whose RPO1-PCR fingerprints were identical to selected RNB and an additional novel isolate. It was found that inoculation of RNB into the pit soils increased and stabilised the RNB population, with MPN comparable to the population in the surrounding undisturbed soils at 4 months post inoculation. So as to maintain the provenance of the RNB population in the SBSLA soils and avoid introducing genetic material that could transfer into the resident RNB, Wattle Grow™ could not be included in the seeding and inoculation trials. In a glasshouse experiment, growth tanks containing pit soil were used to compare the nitrogen fixation efficacy and competitive ability of Wattle Grow™ to nodulate A. ligulata Benth. and A. tetragonophylla F.Muell. against the background RNB in the pit soils and with selected SBSLA isolates over successive sowing periods. No Bradyrhizobium spp. were isolated from the nodules of the Acacia spp. from any of the treatments over the different sowing periods. The majority of the RPO1-PCR fingerprints of the nodule occupants corresponded to SBSLA isolates and an additional three unique fingerprints were identified. The occupancy of the nodules of A. ligulata Benth. and A. tetragonophylla F.Muell. subtly changed with each successive sowing. A number of RNB occurred with greater frequency at the different sowing periods, however, there was a trend towards increased diversity of nodule occupants with each successive sowing, particularly of the RNB nodulating A. tetragonophylla F.Muell.. There was a difference in the response of the two Acacia spp. to the treatments and conditions of the growth tanks. The plant foliage nitrogen concentrations and foliage mass of A. ligulata Benth. were negatively correlated. In contrast, the A. tetragonophylla F.Muell. foliage nitrogen concentrations were positively correlated to the foliage production of these plants. The use of provenant RNB shows potential in improving the germination and establishment of selected legume species in the degraded areas within SBSLA. However, it was shown that different growth conditions for A. ligulata Benth. and A. tetragonophylla F.Muell. alters the symbiotic relationships, nitrogen fixation and growth response of these plants. This illustrates the caution to be exercised when screening for effective symbionts of legumes for the purpose of rehabilitation.

Investigation of the symbiotic associations of Acacia ligulata Benth. and Acacia tetragonophylla F.Muell: The potential for use in the rehabilitation of excavated sites at Shark Bay Salt Pty. Ltd.

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