Nitrogen fixation by subterranean clover in relation to soil acidity and moisture availability

Two of the major constraints to nitrogen fixation and seed production by pasture legumes in the dryland agricultural regions of southern Australia are acid soil infertility and the availability of soil moisture during the growing season. The results presented in this thesis describe the physiological responses of intact plants of subterranean clover (Trifolium subterraneum L.) to the combination of these environmental constraints on clover growth. Measurements of nitrogenase (acetylene reduction) activity using a flow-through gas exchange system were performed initially on a range of pasture legumes important in southern Australian agriculture. This procedure established that a rapid decline in nitrogenase activity and nodulated root respiration occurred within minutes of the introduction of 10 kPa acetylene to the gas stream flowing past the nodules. Based on the respiration coupled to nitrogenase activity and the carbon cost of nitrogenase activity (mol CO2 respired / mol C2H4 produced), calculation of the pre-decline rate of acetylene reduction activity was considered to be the most accurate determination of nitrogenase activity. Using these values, the C2H4 /15N2 conversion factor for the T. subterraneum cv. Seaton Park-Rhizobium trifolii strain WU95 symbiosis was 4.08. However, both the pre-decline rate and magnitude of the acetylene-induced decline in nitrogenase activity decreased during ontogeny. Gaseous diffusion into and out of unstressed subterranean clover nodules was examined at sub- and supra-ambient pO2 and revealed that the permeability of nodules to gases varied over a range of pO2. An increase in the resistance to oxygen flux to the bacteroids at supra-ambient pO2 was postulated to protect nitrogenase from oxygen inactivation and was accompanied by a decline in nitrogenase-linked respiration and hence, nitrogenase activity. As the rhizosphere pO2 declined below 21 kPa, the calculated nodule gas permeability did not alter initially although nodule RQ progressively increased. The resultant decline in nitrogenase activity was consequently due to oxygen deprivation. However, nodule gas permeability increased after 24 h which enabled the maintenance of high rates of nitrogenase activity. In order to more thoroughly study the nitrogen economy of subterranean clover subjected to drought in an acid soil, a glasshouse-based study simulated effects of slow drying of the soil on established plants. This was achieved by basal irrigation of 12 week-old plants grown in cores of (i) undisturbed acid soil (pH 4.3), (ii) cultivated acid soil (pH 4.3) and (iii) cultivated acid soil through which lime had been incorporated to a depth of 12 cm (pH 5.5). In this manner, the soil surrounding the nodules on roots in the uppermost regions of the soil profiles commenced drying. Dehydrating nodules were sensitive to moisture loss and rates of nitrogenase activity declined substantially when nodule water potential (?nod) fell below -0.4 MPa. In contrast, leaf water potential ( ?1) remained relatively independent of ?nod and plant growth rate was maintained due to compensatory nodulation at depth in the moister, less acidic regions of the cultivated soil (with and without the incorporation of lime). Growth rate and accumulation of nitrogen ceased in the undisturbed acid soil as no compensatory nodulation in the extremely acidic subsoil occurred. The characteristics of gaseous diffusion within, and nitrogen fixation by, water-stressed subterranean clover nodules was examined in more detail by passing a drying airstream over the intact nodulated roots. As water and ion uptake by the roots was unimpeded,?nod declined independently of ?1. The decline in specific acetylene reduction activity was most pronounced at a ?nod of -0.4 to -0.5 MPa. This coincided with a large reduction in hydrogen evolution, a pronounced decrease in the respiration coupled to nitrogenase activity and an increase in the carbon cost of nitrogenase activity. Nitrogenase-linked respiration accounted for 50% of nodulated root respiration in unstressed plants but had decreased to 30% of total nodulated root respiration after 8 days of gradual nodule dehydration. Consequently, the calculated nodule diffusion resistance increased continuously, but was most pronounced at a ?nod of -0.4 to -0.5 MPa. Nevertheless, photosynthesis was maintained and soluble carbohydrates accumulated in the shoots, roots and drying nodules. The recovery of nodule respiratory activity following nodule rehydration was due to nodulated root growth and maintenance respiration, not to nitrogenase-linked respiration and the lack of any short-term recovery in acetylene reduction activity was presumed to reflect partial nitrogenase damage. In conclusion, moisture stress resulting in nodule drying readily and irreversibly inhibited nitrogen fixation. In soil, however, growth and nitrogen fixation by subterranean clover were maintained during moderate drought provided that compensatory nodulation could occur at depth in more moist and less acidic subsoil. Leaf water relations were also maintained because water uptake continued whilst the root system was in moist subsoil. In very acid soil, further nodulation was restricted and the mild drought caused premature cessation of nitrogen fixation and growth