Call number: LD2668 .T4 1986 S36 / Master of Science / Horticulture, Forestry, and Recreation Resources

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science Johannesburg, September 2015 / Plant functional traits provide a means to investigate the diverse ecological strategies employed by plants and a tangible link to assess how the variability in these traits might influence ecosystem processes and functioning. The aim of this dissertation has been to determine how plant and soil nutrient dynamics in a savanna environment are affected by two primary drivers, one a top-down driver, being herbivory by large mammalian herbivores and the other a bottom-up driver, the variable N2-fixation capacity of tree species. To the best of my knowledge this is the most comprehensive study to date to investigate the bioavailability of soil nutrients and the link between these availabilities and plant functional traits. Furthermore this study provides important insight into the use of a novel technology, ion exchange resin capsules in a South African savanna context. By studying a selection of plant functional traits (nutrient concentrations, ratios as well as specific leaf area, relative chlorophyll content and leaf dry matter content) and soil nutrients (suite of macro- and micronutrients) associated with two species of savanna tree of contrasting N2-fixation capacities, I went about investigating how herbivory differentially influences the nutrient dynamics of this system. Selecting individuals of the N2-fixing Acacia tortilis and the non-N2-fixing Combretum hereroense both inside an exclosure and on the adjacent land allowed me to determine the potential impacts by herbivores. These include both direct impacts from foraging and indirect impacts through the regulation of nutrient input pathways via deposition of dung and urine. The work compiled for this dissertation is based on the experimental work conducted in a mesic savanna system in the Marakele Park (PTY) Ltd. During the course of this dissertation, I investigated herbaceous and woody biomass in relation to protection from and exposure to herbivory, determining any differences in the functional leaf traits between individuals inside and outside the exclosure, if these differences were exhibited in the associated herbaceous biomass as well as a comprehensive assessment of the bioavailability of 15 important micro- and macronutrients using ion exchange resin capsules. These capsules were incubated in the soil over the entire summer rainfall period, providing a cumulative view of nutrient bioavailability during the growing season. In this work I also demonstrated whether particular nutrients are associated with specific drivers (i.e. herbivory, canopy position or N2-fixation). Furthermore, these results were then looked at together to suggest the mechanism by which herbivory and N2-fixation drive nutrient dynamics and make recommendations on the use of these results in managing savanna systems in the future. Between the two sites, aboveground herbaceous biomass was significantly greater when protected from herbivores than on the adjacent land. Both exposure to herbivory and N2-fixation capacity were found to alter plant functional traits. Herbivore presence was associated with an increase in herbivore-resistant or structural traits such as C/N, C/P, foliar C and SLA as well as a reduction in N and P content. These less palatable leaves were accompanied by a significantly lower availability of a number of important soil elements, namely NO3-N, inorganic N, P, K, Na, Cu, B, Mg, and S. This suggests a feedback loop between these two components of the ecosystem. N2-fixation capacity is associated with greater concentrations of elements such as N and P and a reduction in traits that are illustrative of a greater structural investment into leaves. Soil nutrient bioavailability however, shows a reduction in certain nutrients when associated with Acacia. A number of nutrients which show a reduction in availability are those which are essential to N2-fixation machinery, namely B and Fe but also lower bioavailabilities of Al and Mg. Finally, Ca, NO3-N, B, Fe, Al and inorganic N were found in greater quantities below the tree canopy than beyond it. In conclusion both herbivory by large mammalian herbivores and N2-fixation have significant effects on tree health, through their regulation of limiting nutrients and alteration of leaf traits. Given the changes which these drivers are capable of exerting on plant and soil nutrient dynamics, this has important consequences for ecosystem processes and functioning and highlights potential considerations in the long-term sustainable management of savannas.

The rhizobia-legume symbiosis is the most agriculturally significant source of naturally fixed nitrogen, accounting for almost 25% of all biologically available nitrogen. Rhizobia-legume compatibility restrictions impose limits on symbiotic nitrogen fixation. In many cases, the molecular basis for symbiotic compatibility is not fully understood. The signals required for establishing a symbiotic partnership between nitrogen-fixing bacteria (e.g. Sinorhizobium meliloti) and leguminous plants (e.g. Medicago truncatula) have been partially characterized at the molecular level. The first stage of successful root colonization is competitive occupation of the rhizosphere (which is poorly understood). Here, the bacteria introduce themselves as potential symbiotic partners through the secretion of glycolipid "Nod" factors. In response, the host facilitates a more exclusive mode of colonization by the formation of a root nodule – a new organ capable of hosting dense intracellular populations of symbiotic rhizobia for nitrogen fixation. This dissertation reports the exhaustive identification of S. meliloti genes that permit competitive colonization of the M. truncatula rhizosphere, and includes a mechanistic study of one particular bacterial signaling pathway that is crucial for both rhizosphere colonization and nodulation. I have made use of Tn-seq technology, which relies on deep sequencing of large transposon mutant libraries to monitor S. meliloti genotypes that increase or decrease in relative abundance after competition in the rhizosphere. This work included the collaborative development of a new computational pipeline for performing Tn-seq analysis. Our analysis implicates a large ensemble of bacterial genes and pathways promoting rhizosphere colonization, provides hints about how the host plant shapes this environment, and opens the door for mechanistic studies about how changes in the rhizosphere are sensed and interpreted by the microbial community. Notable among these sensory pathways is a three-protein signaling system, consisting of FeuQ, FeuP, and FeuN, which are important for both rhizosphere colonization and nodule invasion by S. meliloti. The membrane-bound sensor kinase FeuQ can either positively or negatively influence downstream transcription of target genes by modulating the phosphorylation state of the transcriptional activator FeuP. FeuN, a small periplasmic protein, inhibits the positive mode of FeuPQ signaling by its direct interaction with the extracellular region of FeuQ. FeuN is essential for S. meliloti viability, underscoring the vital importance of controlling the activity of downstream genes. In summary, I have employed several powerful genetic, genomic, computational, and biochemical approaches to uncover a network of genes and pathways that coordinate root colonization and nodulation functions.

Biological nitrogen fixation is accomplished in the bacterium Azotobacter vinelandii by means of three metalloenzymes: The molybdenum, vanadium, and iron-only nitrogenase. The knowledge regarding biological nitrogen fixation has come from studies on the Mo-dependent reaction. However, the V- and Fe-only-dependent reduction of nitrogen remains largely unknown. By using homology modeling techniques, the protein folds that contain the metal cluster active sites for the V- and Fe-only nitrogenases were constructed. The models uncovered similarities and differences existing among the nitrogenases regarding the identity of the amino acid residues lining pivotal structural features for the correct functioning of the proteins. These differences, could account for the differences in catalytic properties depicted by these enzymes. The quaternary structure of the dinitrogenases also differs. Such component in the Mo-nitrogenase is an α2β2 tetramer while for the V- an Fe-only nitrogenase is an α2β2δ2 hexamer. The latter enzymes are unable to reduce N2 in the absence of a functional δ subunit, yet they reduce H+ and the non-physiological substrate C2H2. Therefore, the δ subunit is essential for V- and Fe-only dependent nitrogen fixation by a mechanism that still remains unknown. In attempt to understand why the δ subunit is essential for V-dependent N2 reduction from a structural stand point, this work presents the strategy followed to clone the vnfG gene and purify its expression product, the δ subunit. The purified protein was subjected to crystallization trials and used to stabilize a histidine-tagged VFe protein that would otherwise purify with low Fe2+ content and poor H+ and C2H2 reduction activities. The VFe preparation was used to conduct substrate reduction assays to assess: i) The electron allocation patterns to each of the reduction products of the substrates C2H2, N2, N2H4, and N3−; and ii) Inhibition patterns among substrate and inhibitor of the nitrogenase reaction. This work also reports on the effect N2H4 and N3− has on the electron flux to the products of the C2H2 reduction. The work presented herein provides information with which to compare and contrast biological nitrogen fixation as catalyzed by the Mo- and V-nitrogenases from Azotobacter vinelandii.

The inputs and losses of nitrogen from Great Basin arid soils were studied using the acetylene reduction and 15N techniques. Filamentous blue-green algae were observed to be the predominant algal group in the soil crusts. The bacterial association with this group of algae suggest a phycosphere-like effect, thus allowing heterotrophic nitrogen fixation and denitrification to occur. Up to 17.5 mg N/100 g soil was found to have been fixed in surface soils (0 to 3 em) during a three week incubation period, while 45.9 mg N/100 g soil was fixed in a five week incubation period. Ammonium sulfate and ammonium sulfate plus plant material amendments reduced the gain in nitrogen by 41 to 100 percent. 15 + 15 - Fifty to sixty percent of the applied NH4-N and N0 3-N was denitrified during the first week of incubation while 70 to 80 percent of the NH 4-N was lost in a three- to five-week incubation period. These data suggest that a potential for heterotrophic nitrogen fixation exists, and under optimal conditions, significant gains in soil nitrogen may be achieved. However, in the presence of allelochemic agents, the potential gain in soil nitrogen may be reduced or inhibited. In addition, the denitrification potentials of these soils may also limit the input of nitrogen. The application of protein ( casein) to these soils resulted in an ammonification rate of 50 to 60 percent. 15 Fixed N2 indicated a 21 to 48.8 percent ammonification rate, thus suggesting that the mineraliztion of NH 4 was the rate limiting step for nitrogen loss. Ammonia volatilization accounted for less than a five percent nitrogen loss, regardless of experimental conditions. The inhibitory effects of plant material and litter extracts, and ''N-Serve" on heterotrophic nitrogen fixation has been assessed. The data suggest that the nitrogen fixing population is sensitive to the inhibitory effects of such agents .

The rhizobium – legume symbiosis is a complex process that involves genetic cooperation from both bacteria and plants. Previously, our lab described naturally occurring accessory plasmids in rhizobia that inhibit this cooperation. A transposon mutagenesis was performed on the plasmids to detect the genetic factor that blocked nitrogen fixation. Several of the plasmids were found to possess a replication operon that when disrupted by transposon insertion, restored symbiotic function. This study describes an in-depth investigation into one of those plasmids, pHRC377, and into its replication operon. The operon, which we have called repA2C2, comes from the repABC family of replication and partitioning systems commonly found in alphaproteobacteria. In this study we show that this operon is not necessary for pHRC377 replication in LB culture or free living cells, but is necessary for plasmid amplification in the plant, specifically during rhizobial differentiation into nitrogen fixing bacteroids. We also show how the other repABC type operons on pHRC377 function in relation to plasmid maintenance and copy number during endoreduplication and how they do not have the same phenotypic effect as repA2C2.

Trifolium ambiguum (Caucasian clover) is being released in New Zealand for use in areas where growth of T. repens (white clover) is marginal. Although closely related to T. repens, T. ambiguum has unique and highly specific nodulation requirements and as rhizobial strains capable of effectively nodulating T. ambiguum are not naturally found in New Zealand soils, they must be introduced with the seed. Rhizobium leguminosarum bv. trifolii strains such as ICC105 form effective nodules on T. ambiguum but ineffective (Fix⁻) nodules on T. repens. The T. repens nodules nevertheless develop normally and contain bacteroids. R. l. bv. trifolii strains that are effective on T. repens such as NZP561, fail to nodulate T. ambiguum. As the host-specific nitrogen fixation defect of Caucasian clover rhizobia on T. repens has potentially adverse agronomic implications, the genetic basis for this Fix⁻ phenotype was investigated. Rhizobium leguminosarum bv. trifolii strain ICC105 was converted to Fix⁺ on T. repens by the introduction of an 18-kb fragment of DNA from a white clover rhizobial strain (NZP514) symbiotic plasmid. This fragment contained several nif and fix genes, including nifHDKEN, fixABCX, nifA, nifB, fdxN and fixU. Tn5 mutation of these white clover rhizobial genes demonstrated that most were required to impart the Fix⁺ phenotype on T. repens to ICC105, with the exception of nifA. Mutagenesis of the ICC105 nifA gene and subsequent complementation with various combinations of the white clover rhizobia nif/fix genes as well as transcriptional lacZ fusion studies of the ICC105 nifA and nifH genes demonstrated that ICC105 nifA is expressed and functional during the ineffective nodulation of T. repens and able to activate expression of nifHDKEN and fixABCX operons derived from white clover rhizobium but not from ICC105. Sequence analysis and comparison of the intergenic region between the divergently transcribed nif/fix operons revealed a conserved 111-bp region found between the nifH/fixA promoters of Caucasian clover rhizobia, but not in white clover rhizobia. Attempts to modify this region in ICC105 failed in creating a strain which was Fix⁺ on T. repens; however recombination of the nifHD/fixAB region from a white clover rhizobium into the ICC105 genome produced several strains with a �swapped� nitrogen fixation phenotype (i.e. Fix⁺ on T. repens and Fix⁻ on T. ambiguum). A hypothesis was therefore proposed by which differences in the nifH/fixA promoter regions of Caucasian clover rhizobia and white clover rhizobia modulate the expression of the upstream genes in response to the particular plant host they are nodulating. The incompatibility between the symbiotic plasmid of R. l. bv. trifolii ICC105 and the white clover rhizobium symbiotic plasmid cointegrate, pPN1, was also investigated and potential regions of each plasmid involved in this incompatibility were identified. The research presented in this thesis has contributed to the genetic knowledge of the nitrogen fixation genes, and regulation of these genes in R. l. bv. trifolii. It has also provided progress towards the goal of creating a suitable inoculant strain for T. ambiguum that is able to fix nitrogen in symbiosis with both T. repens and T. ambiguum.

Bibliography: leaves 189-210 / xxxiv, 210 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agricultural Biochemistry, 1987

Bibliography: leaves 153-179. / xvi, 179 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Annual species of Medicago, or medics, are important pasture legumes in the neutral to alkaline soils of southern Australia but their nodulation and nitrogen fixation processes are retarded by soil nitrate. This study ascertains whether an observed tolerance to nitrate among medic species can be substantiated, and attempts to understand the underlying factors responsible. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, (1995)

Bibliography: leaves 153-179. Annual species of Medicago, or medics, are important pasture legumes in the neutral to alkaline soils of southern Australia but their nodulation and nitrogen fixation processes are retarded by soil nitrate. This study ascertains whether an observed tolerance to nitrate among medic species can be substantiated, and attempts to understand the underlying factors responsible.