Novel approaches to manipulating proteolysis in ensiled perennial ryegrass (PRG) were investigated. The effects of the following on nitrogen (N) distribution in silage were investigated: E- 64, a specific cysteine-peptidase inhibitor (CPI); pepstatin A a specific aspartic-peptidase inhibitor; cystamine dihyrochloride (CYS) and N-ethylmaleimide, general CPIs, and formic acid (FA). Treatment with FA or CPIs reduced total soluble N, as a result of reduced proteolysis, and FA and CYS treatments increased peptide N concentrations (determined using fluroescamine or ninhydrin assays, and by amino acid analysis) compared to the control. Pepstatin A had little or no effect on the N constituents of silage. Characterisation of silage peptides using Sephadex G-25 suggested that they were predominantly di and tripeptides, with a small proportion of longer peptides (>7 amino acid residues). Forty additional compounds were screened for their efficacy as inhibitors of proteolysis in aqueous extracts of PRG. Five selected compounds were applied to PRG at ensilage: TPCK, a non specific CPI; chelators, 1,10-phenanthroline and 8-hydroxyquinoline (8-HQ); bestatin, a metallo-peptidase inhibitor; and N-acetyl-L-tyrosine ethyl ester (ATEE), a serine-peptidase inhibitor. When compared to the control, TPCK and 1,10- phenanthroline reduced total soluble N and increased peptide N concentrations; 8-HQ increased only peptide N concentrations. These chelators also restricted fermentation. The effects of Trypticase (peptides produced by enzymic hydrolysis of casein), silage extracts and N fractions prepared from silage extracts by cation exchange chromatography, as sources of N, on the growth of rumen bacteria, <I>Megasphaera elsdenii, Prevotella ruminicola</I> and <I>Selenomonas ruminantium, </I>supplied with glucose as an energy substrate <I>in vitro, </I>were investigated. No growth was observed on media containing extracts from silages produced in the presence of chelators but all bacteria grew on purified N fractions. Increasing silage peptide N therefore did not enhance microbial growth but for some treatments, silage N supported faster growth than Trypticase.

The microbiology of nitrification has been extensively studied, but the ecology and environmental impact of the process has received less attention. The reason for this has more to do with the difficulties of conducting field experiments to examine the links with other processes which cause losses of nitrogen, than a failure to appreciate its importance in agricultural systems. This project was designed to overcome some of the limitations of existing field techniques to enable simultaneous measurements of nitrification and the major processes of N supply (mineralization) and N loss (denitrification and leaching) to be examined. The study proceeded in three distinct phases: firstly, soils with contrasting N management histories were examined, using laboratory assays for potential activities. Clear differences which resulted from higher N inputs were established, with correspondingly higher nitrifying activities. For example, in a fertilized soil, mmoniaoxidizers produced 48.4 compared with 1.3 nM NO2- g-1 soil h-1 in an unfertilized soil. Potential nitrite-oxidizing rates were 93.4 and 62.5 nM g-1 h-1, respectively. Assays of enzyme kinetics, therefore confirmed the higher nitrifying activity in the fertilized soil, but demonstrated a lower affinity of the enzyme for N02 substrate, with Km values of 436 and 310 gM NO2--N, respectively. Nitrifying rates in soils from grass-clover swards were intermediate between the fertilized and unfertilized soils. Secondly, a new field incubation technique was developed and used to obtain actual rates by concurrent measurements of the major N cycling processes. A strong correlation was established between nitrification and denitrification (r2 = 0.98). The measurements were verified by comparison with other independent methods. Net rates of nitrification in the same soil type ranged from 0.55 - 1.17 kg N ha-1 d-1, with the highest rates in the fertilized soil. Over 70% of the mineralized N was nitrified, of which 80% was subsequently lost (i. e. either denitrified or leached). Thirdly, the practical implications of these findings were examined in greater detail using 15N labelling techniques which enabled process rates (net and gross) to be established in a model of the N cycle. When nitrification was inhibited, there were no significant differences between gross or net mineralization rates in the soils from the three swards, which indicated that N-immobilization could be directly influenced by the level of nitrifying activity in these soils. The influence of nitrification in determining the pathways of N loss from grassland soils was quantified in this study. From a detailed investigation of the processes involved in N cycling, it was deduced that nitrification was also one of the major factors in determining the outcome of competition for inorganic N between plant and microbial biomasses.

The effect of foliar applied potassium chloride on Septoria tritici on winter wheat was quantified and possible modes of action investigated by in vitro, glasshouse, controlled environment and field experiments between 1996 and 1999. In vitro experiments showed that as the concentration of potassium chloride increased, mycelial growth and germination of conidia decreased (EO sos of 1.36M and 0.7M, respectively). One glasshouse and two field experiments showed a significant reduction in the leaf area affected by S. trttict after foliar application of potassium chloride compared to untreated controls. In general, application of potassium chloride reduced the leaf area affected by S. tritici by 20 -.40%. However, a significant yield increase was not observed. Potassium chloride applied to the lower leaves of winter wheat did not confer systemic acquired resistance against S. tritici on the upper leaves. Inhibition of conidial germination, on leaf surfaces by potassium chloride was observed. Similar inhibition was observed when polyethylene glycol, an inert osmoticum, was applied at the same calculated osmotic potential. During field experiments there was no significant difference in the leaf area affected by S. tritici in plots treated with potassium chloride or polyethylene glycol, although both significantly reduced the leaf area affected compared to untreated control plots. Therefore, the principal mode of action of potassium chloride was proposed to be as a result of adverse osmotic conditions caused by the salt on leaf surfaces. However, the addition of a range of adjuvants to potassium chloride did not increase the efficacy of S. tritici control in glasshouse studies. The results from this study show that potassium chloride, when applied to foliage of winter wheat can reduce the leaf area affected by S. tritici and it is proposed that this reduction was by adverse osmotic conditions caused by the salt on leaf surfaces.

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Two groups of soils were studied, the first being made up of twelve heavily fertilised representative soils from the European Community and the second containing fertilised and unfertilised soils for comparison. The first group demonstrate how the amount of plant available P was generally most closely related to soil P content, rather than soil chemical characteristics. However, the total adsorption capacity tends to decrease in the order moderately acid > calcareous > weakly acid. A very steep increase in solution P concentration above 60 percent saturation was observed and it was calculated that 15 to 30 years of cropping without further fertiliser P additions is required to bring some soils below this level. The second group of soils shows how long term P fertiliser additions cause accumulation of all forms of inorganic P. The comparison of a continuously fertilised soil to one which has had no P additions for three years, suggests that once soils of this type have been fertilised in excess of plant requirements, they can be cropped for several years without decreasing yield due to P deficiency. The aggregate separation indicates how selective erosion of small aggregates leads to increased loss of P in most cases. Mixing of aggregates demonstrated how some have a stronger ability to influence concentration than others and this was linked to their phosphate buffer capacity. Reducing conditions increased P release and this was linked to a combination of soil type, P content and cation release. Fertilisation has increased the available P and the ability to influence solution P most in the fraction at the greatest risk from erosion.

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