Sustainable reintroduction of the nitrogen cycle post coal mining utilizing the legume-rhizobia symbiosis

Research Doctorate - Doctor of Philosophy (PhD)Mining in general, and open cut mining in particular, is an inherently destructive process. Mining occupies a very small area of the Australian continent, but causes intense disruption to the environment when concentrated in relatively small regions where minerals outcrop, or are close to the surface. In the upper Hunter River Valley region of New South Wales, open cut coal mining currently directly covers 500 km² or 1/6 of the upper valley. Revegetation strategies have moved from rehabilitation to past land use, usually grazing in the Hunter Valley, to reconstruction of native ecosystems, whether wooded or native grasslands. At the Mount Owen mine, mining consent requires reconstruction of approximately 220 ha of the Ravensworth State Forest, a dry sclerophyll forest ecosystem dominated by trees from the genus Eucalyptus. Additionally the mine owner and operator have decided to revegetate to forest or woodland the entire disturbance area of about 960 ha. Topsoil transfer is the usual method of re-establishing native vegetation, but forest topsoil supply is inadequate to fulfil the stated goals. Spoil, the crushed rock from just below the soil horizons down to, and between, the coal seams, is not a medium conducive to plant growth. It is biologically dead, deficient in nitrogen and other plant available nutrients and not having been subjected to soil building processes, lacks any structure. To support a native forest ecosystem, with all its constituent vegetation layers, spoil needs to be physically amended with soil replacement media, preferably forest topsoil, but other materials such as pasture subsoil, chitter (coal washing waste) and, or biosolids (dried sewerage sludge). Nutrients added as fertilizers have been the traditional method of overcoming soil nutrient deficiencies. But this is an expensive, inefficient practice that can result in pollution from leaching and runoff. One method that can be used to circumvent these problems is the addition of soil microbes that mediate the input of plant available nutrients from the soil medium. As nitrogen has been identified as being particularly deficient in this system, the re-introduction of native legumes with their rhizobial symbionts was seen as a sustainable method of supplying nitrogen to the reconstructed forest ecosystem. To this end, root nodules from native legumes growing in the Ravensworth State Forest, Mount Owen mine rehabilitation areas and Donaldson mine, further down the valley, were collected, sterilized and cultured. Thirty-four bacterial cultures were subjected to culture-dependent and molecular identification. Twenty-three isolates were confirmed as nodulators by their ability to re-nodulate Macroptilium atropurpureum. Using PCR and targeting a 500bp fragment of the 16s rRNA gene seven members of the genus Rhizobium were identified, including three of the same strain, and two from the genus Agrobacterium. Seven isolates were identified as not having been previously described. Three of these exhibited high homology to the genus Pseudomonas and four were identified from the genus Burkholderia. The isolate that was chosen for pot trials and the field inoculum was later identified as belonging to the genus Burkholderia, which at the time of identification had not been previously reported in Australia. These nodulating cultures were inoculated onto seedlings of six legumes that were to be used in the revegetation efforts at Mount Owen mine and tested for specificity and effectiveness in promoting growth in a series of controlled environment trials. The legume species included the dominant middle storey small tree, Acacia parvipinnula, four shrubs, Indigofera australis, Daviesia ulicifolia, Acacia amblygona, Pultenaea retusa, and a creeper or twiner, Hardenbergia violacea. Specificity was determined by the ability to nodulate, with functional nodules scored by observation of a rust red colour indicating the presence of leghaemoglobin. Effectiveness of growth promotion was determined by shoot dry weight and its ratio with nodule dry weight. Results showed marked variation in host-symbiont specificity and significant differences in the ability to promote growth of the legume hosts. A. parvipinnula proved to be the least specific host (nodulated by 22/23), while D. ulicifolia (nodulated by 13/23) and P. retusa (nodulated by 8/23) were the most specific. Bacteria also displayed specificity with six isolates able to form functional nodules on all six hosts. The remaining isolates were either unable to nodulate all of the hosts or formed non-functional nodules. No single isolate proved to be a highly effective growth promoter on all host legumes. For reasons of expediency in progressing the project, the first isolate that formed functional nodules on all hosts was chosen as the field trial inoculum. The ability of the selected inoculating isolate to promote the growth of a simulated community of plants, including the six test legumes used for specificity testing, found in the Ravensworth State Forest when grown in a range of soil replacement media was then tested in shade house and field trials. The shade house trial demonstrated the ability of the inoculum to successfully increase the growth of the plant community, including in media with a resident rhizobia population, one potential obstacle to successful inoculum establishment. Under field conditions at the Mount Owen mine, inoculation of the six host legumes with the selected isolate successfully increased survival and dry weight of five of the species, albeit with some interaction between host legumes and soil replacement media. In conclusion, a number of new soil dwelling bacteria have been cultured and warrant further investigation, including a novel strain of nodulating Burkholderia. It was demonstrated that specificity exits between the native legumes and nodulating symbionts tested, with significant differences in ability to nodulate and promote plant growth. Further testing of specificity for other commonly found legumes is warranted. Investigation of the efficacy of multi-isolate inocula versus single isolate inoculum trialled here to increase effectiveness and to facilitate the re-introduction of native nodulators is also suggested. Successful inoculation was shown to enhance revegetation efforts by increasing survival and plant growth in a range of soil replacement media