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In this study, six annual legumes and the perennial, red clover (Trifolium pratense L.) were monocropped (MC) and intercropped (IC) with barley in a field study with three N levels, 0, 30 and 60 kg N ha$ sp{-1}$. At O kg N ha$ sp{-1}$, N$ sb2$-fixation and N transfer were estimated by the $ sp{15}$N isotope dilution (ID) method. At 60 kg N ha$ sp{-1}$, a direct $ sp{15}$N labelling method was employed to study N transfer. The hypotheses were that the annual species would be more productive within one growing season than red clover, that increased N levels would increase herbage dry matter (DM) and crude protein (CP), that the proportion of N derived from N$ sb2$-fixation in IC-legumes would be higher than that of MC-legumes and that within intercrops there would be evidence of N transfer. In neither year was the total DM yield of red clover, MC or IC, less than the rest of the legumes. In 1991, the total DM yield of intercrops responded to 30 kg N ha$ sp{-1}$; in neither year did the estimated total CP yield of MC-legumes or intercrops respond to N levels. Only in 1992 was there evidence of N$ sb2$-fixation and the proportion of N derived from fixation by IC-legumes was 145% higher than that of MC-legumes. Only the $ sp{15}$N direct labelling method gave evidence of N transfer, to associated legume and barley plants in 1991, and to associated legume plants in 1992.

This research was conducted at the University of KwaZulu-Natal, Pietermaritzburg, South Africa. The research encompasses five different studies to assess nutrient cycling in intensive and extensive grazing systems with a view to optimising livestock production. The first study was designed to assess the effect of teff-lucerne mixtures on teff, lucerne and teff-lucerne mixture yields. Lucerne and teff-lucerne mixtures benefited from the association. The overall soil N content of the teff-lucerne mixture plots was greater than the teff alone plots. The second study focused on teff-leucaena association evaluation. It had two leucaena plant row spacings as treatments, 180cm and 120cm, respectively. Teff grown in mixture with leucaena produced a total teff dry matter (DM) of 7931.57 kg ha¯¹ for the 180cm row spacing and 8329.57 for the 120cm row spacing compared to the 3548.93 kg ha¯¹ of DM obtained from the teff alone treatment. The teff-Ieucaena stand also had a greater DM yield response to leucaena row spacing compared to the teff alone. In terms of nutritive quality, all stands from the teff-leucaena plots were better than the quality obtained from the teff alone plots. Total N content of teff from the l80cm row spacing was 21.83 g kg¯¹ and that from the 120cm 16.07 g kg¯¹ compared to the total nitrogen (N) content of 19.77 g kg¯¹ of the teff alone treatment. The total phosphorus (P) content was 2.73, 1.96 and 2.07 g kg¯¹ for the 180cm, 120cm and teff alone treatments respectively. However, the total soil N content was higher for the teff alone plot than for the teff-leucaena plots, which are 1.91, 1.48 and 100 g kg¯¹ for the teff alone, 180cm and 120cm treatments respectively. The third study was designed to assess the effects of different N fertilizer application rates on teff yield response. The rates applied were 0, 50, 100 and 150 kg N ha¯¹. There was significant difference in teff response of the three N fertilizer application rates compared to the control and teff DM yield response was lower for the 150 kg N ha¯¹ (838 kg ha¯¹) treatment compared to the control (553 kg ha¯¹). Both teff DM and nutritive value were higher in the plots treated with N fertilizer than in the plot which received no N fertilizer (control). The soil N content was also higher in those plots treated with N fertilizer. Study four was conducted on the Department of Grassland Science's grassland management techniques trial field at Ukulinga. The effects of nutrient cycling under different management techniques such as burning, mowing and grazing on grass yield response, plant quality and soil nutrients were assessed. However, the response of grass DM yield and P content was not significant but the three treatments had a significant effect on grass N content. Their effect on soil N content was also significant and the grazing plot had the greatest soil N levels. The last study was conducted in the rural areas of Okhombe and Zwelitsha to assess the effects of grazing intensity on grass yield response, plant quality and soil nutrient status at different distances from homesteads. Grass DM yield and nutritive value declined when distance from the homestead increased. The soil N content also was higher nearer to the homestead than further away. Most farmers, particularly in developing countries including those in Eritrea, often experience that their animals prefer forages from some plants such as lucerne, leucaena, and other indigenous leguminous plants. They also observe that they get greater yield from crops grown near leguminous plants or in rotation with legumes. They are also still using manure from their animals to fertilize their croplands. Therefore, it is still the duty of the researchers to demonstrate to farmers on farm studies to convince farmers that it is because leguminous plants have the ability to add quality and quantity to the feed of the animals and soil nutrients to the croplands. Hopefully, this study will convey to farmers the use of growing integrated grassllegume pastures and crops, and illustrate that livestock have their own role in transporting nutrients and hence use them as good means of distributors of soil nutrients. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Ab einem Preisniveau von 2 Euro/kg für industriell hergestellten Stickstoff wird der Leguminosenanbau durch die biologische Stickstofffixierung in der konventionellen Landwirtschaft wirtschaftlich vorteilhaft. Körnermais und teilweise Winterraps können dann von Ackerbohnen aus den Fruchtfolgen verdrängt werden. Dies wurde mittels Linearer Programmierung für einen durchschnittlichen landwirtschaftlichen Betrieb in Sachsen errechnet. Kleearten und Luzerne als Futterpflanzen werden unter diesem Blickwinkel aber erst ab Kosten von 2,30 Euro/kg Industriestickstoff konkurrenzfähig gegenüber Silomais. Letztlich entscheiden vor allem die Preis-Kosten-Relationen von Betriebsmitteln und Agrarerzeugnissen über den Einsatz der biologischen Stickstoffgewinnung. Dazu wurden zwei Szenarien dargestellt. Nicht zu erwarten ist, dass stark steigende Preise für Mineralstickstoffdünger in Zukunft zur vermehrten Umstellung auf ökologischen Landbau führen werden, einem Anbausystem, das sich fast vollständig auf die legume Stickstoffbindung stützt.

Legume-rhizobia symbioses contribute at least 20% of the biosphere’s supply of reactive nitrogen. These unique associations rely on the exchange of specific molecular signals between nitrogen-fixing soil bacteria, collectively called rhizobia, and their host plants and, with few exceptions, result in the formation of root nodules, which provide an environment suitable for nitrogen fixation. However, nitrogen fixation is energetically expensive and nodule proliferation, in much the same manner as the proliferation of other meristems in plants, must be controlled in order to attain equilibrium between cell proliferation and differentiation. Nodule proliferation is controlled primarily and systemically by autoregulation of nodulation (AON). A gene central to this process was first isolated by map-based cloning from soybean (Glycine max) and was named G. max Nodule Autoregulation Receptor Kinase (GmNARK) in accordance with its biochemical and physiological functions. Expression patterns of GmNARK have been described by several investigators; however, these reports were based on either non-quantitative methods or a limited number of tissue types. More importantly, the expression domains of GmNARK were completely unknown. The study described in this thesis utilised techniques such as quantitative RT-PCR (QRTPCR), transcription start site mapping, promoter-reporter gene fusion, and promoter deletion, to analyse the expression levels and domains of GmNARK across a variety of tissues as well as identify the promoter elements that are responsible for the basal and tissue-specific expression of GmNARK. In addition, the promoter activity of GmNARK was also compared with that of Lotus japonicus HAR1, the GmNARK orthologue, in both homologous and heterologous transformation systems. Based on QRT-PCR, GmNARK was expressed to varying levels throughout the plant; the transcript was detected at high levels in mature leaves and roots but to a lesser extent in young leaves, shoot tips and nodules. The transcript level was not significantly affected by Bradyrhizobium japonicum during the first week following inoculation. Histochemical analysis of L. japonicus plants carrying either a 1.7 kb GmNARK promoter or 2.0 kb LjHAR1 promoter fused to a -glucuronidase reporter gene localised GUS activity to living cells within vascular bundles, especially phloem cells in leaves, stems, roots, and nodules. Phloem-specific expression was also detected in soybean hairy roots carrying these constructs. These results suggested that both cis- and trans-acting elements required for the transcriptional regulation of these orthologous genes are likely to be conserved. In contrast, 1.7 kb of the GmNARK promoter did not drive phloem-specific expression in Arabidopsis thaliana, indicating the absence of the trans-acting elements required for the tissue-specificity of GmNARK in this distantly related species. The comparison of 2.0 kb of promoter sequences of GmNARK, LjHAR1 and Medicago truncatula SUNN, another GmNARK orthologue, using bioinformatics and computational approaches indicated several highly conserved motifs including a putative negative regulatory region (NRR), which was previously reported to repress gene expression in non-phloem cell types. Deletion analysis of the GmNARK promoter, however, ruled out the possibility that this motif, found at -308 bp with respect to the translation start site, was truly functional and located the region controlling phloem-specific expression to DNA sequence between 908 bp and 1.7 kb upstream of the start codon. Two other candidate regions were identified by Multiple EM for Motif Elicitation (MEME). These regions, namely MEME3 and MEME4 showed strong sequence similarity to the corresponding regions of the LjHAR1 promoter. Interestingly, the MEME3 motif was also found in the MtSUNN promoter at a similar location to that of LjHAR1. Potential NRRs in the LjHAR1 and MtSUNN promoters were found in the MEME3 motifs, whereas only a variant form of a NRR in the GmNARK promoter was found in this region. Additionally, an identical semi-palindromic sequence was also observed in the MEME3 motifs of the three orthologous promoters. Based on these findings, the semi-palindromic sequence and the variant form of the NRR are proposed to be positive and negative regulatory elements for the phloem-specific expression of GmNARK, respectively. The computational approaches also identified two potential TATA elements in the GmNARK promoter. Rapid amplification of 5’ cDNA ends and promoter deletion analysis have confirmed that they were functional. The two TATA elements in GmNARK promoter appeared to cooperatively direct transcription of GmNARK, but either was adequate for basal transcription. The finding that the expression of AON receptor-like kinase genes is phloem-specific has contributed to a better understanding of AON signalling pathways. The QRT-PCR study and the discovery of cis-acting regulatory regions have also provided crucial information on the transcriptional regulation of GmNARK as well as plant genes in general. Additionally, the promoters of GmNARK and LjHAR1 could potentially be used to drive phloem-specific expression in legume biotechnology research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

Population dynamics of plants is the study of the recruitment and longevity of plants. Of the many tropical pasture legumes now in use, only Siratro (Macroptilium) is understood in simple mixtures. Walker (1980) found that rapid changes occurred in the populations of some tropical perennial legumes in response to grazing by cattle. The study investigated initial effects of cattle grazing on the dynamics of five perennial, tropical legumes in association with two contrasting grass species.

The fungus Macrophomina phaseolina is the causal agent of several diseases of mungbean (Vigna radiata). One of these diseases, known as charcoal rot, causes spoilage of germinating seed lots, and occurs when seed contaminated with M. phaseolina is used for sprouting. The detection of the pathogen during seed testing prior to export leads to downgrading of the seed and a resultant financial penalty to the grain grower. The aims of this research were: to determine the location of M. phaseolina in diseased and symptomless tissue of mungbean plants; to determine the mode, site and timing of seed colonisation of mungbean; to determine the impact of biotic and abiotic factors on seed colonisation; to conduct an assessment of genotypic variation of M. phaseolina within single plants; to determine modes of transport of inoculum that contribute to foliage infection and seed colonisation of mungbean; and to assess both pre- and post-harvest management strategies in order to reduce or prevent seed colonisation, or to minimise the process of seed transmission. The location of M. phaseolina in mungbean plants was determined through serial sectioning of, and subsequent isolation from, naturally infected host tissue. A large proportion of the mungbean tissue infected by M. phaseolina was found to be symptomless. Moreover, there were large areas of ostensibly pathogen-free tissue separating infection foci, thus indicating a strong likelihood of independent aerial infections. A selection of 14 isolates obtained from serial sectioning were assessed for genotypic variation with six primers using RAPD analysis. Of the 36 bands that were scored, 78% were polymorphic and as a result, 12 distinct genotypes were detected. Polymorphisms were also detected amongst isolates obtained from the same discrete infection area on single plants, which strongly suggests the occurrence of multiple aerial infections. Various methods of controlled inoculation including soil infestation, pod and foliar inoculations, and artificial seed infestation, were used to determine how mungbean seeds are colonised by M. phaseolina. Additionally, most of these inoculation methods were coupled with a series of abiotic treatments (temperature regimes, watering regimes, application of herbicides) that were designed to initiate stress conditions within infected plants, and possibly trigger growth of M. phaseolina from the infection courts and colonise seed. Seed colonisation was established in vitro and in vivo when immature pods were directly inoculated with microsclerotia of M. phaseolina. At least two days exposure at 100% relative humidity (RH) was necessary to establish seed infection in detached mungbean pods that were inoculated with microsclerotia of M. phaseolina. Extensive seed infection was still obtained when one or more days of 100% RH was interrupted by up to three days at low humidity. All except one of the other methods of controlled inoculation failed to produce colonised seed even when combinations of stresses were applied. Only when the bipyridylium herbicide ‘Spray Seed 250’ was applied to plants following the inoculation of mungbean stems within 13 cm of the pods, was seed colonised by M. phaseolina. This result raises the possibility that delayed harvesting of desiccated mungbean crops may promote further colonisation of mungbean tissue, including seed. Very strong evidence for the colonisation of mungbean seeds after deposition of soil-splashed inoculum of M. phaseolina onto pods was obtained through field and laboratory-based studies. Soil-splashed inoculum (most likely microsclerotia) was also found to be the source of inoculum responsible for the development of Macrophomina leaf blight of mungbean at several regional sites. To further investigate this finding, areas of several mungbean crops growing in naturally infested soil were covered in hessian cloth to prevent soil-splash and assessments of the levels of seed colonisation between covered and uncovered areas were made. Although colonisation of seed in the covered areas was significantly lower than in the uncovered areas, covering the soil did not eliminate colonisation of seed. This result suggests that inoculum dispersal, leading to pod infection and subsequent seed colonisation, occurs not only in splashed-soil but also by other means. Soil transported to the extra-floral nectaries of mungbeans by ants was found to contain infective inoculum of M. phaseolina. Furthermore, air-borne debris and dust collected in a trap contained viable microsclerotia of the pathogen. Isolates of M. phaseolina collected from both sources were pathogenic on mungbean seedlings, and suspensions of ant-transported soil and air-borne dust/debris infected mungbean pods and seed. This is the first report of both modes of inoculum dispersal. All three modes undoubtedly contribute to the total level of colonised seed, but their relative importance remains to be determined. Several options for the management of charcoal rot in mungbean seeds were investigated. Application of the fungicide carbendazim to mungbean plants after flowering significantly decreased, but did not prevent, colonisation of mungbean seed by M. phaseolina. Consequently, this method of management holds little promise for mungbean growers. A large number of weeds common in Australian mungbean fields were newly reported as hosts of M. phaseolina. Isolates obtained from the infected, but symptomless weeds were pathogenic on mungbean seedlings, thus indicating a lack of host-specificity toward mungbean. It is strongly suspected that the use of herbicides to control weeds in reduced and zero-tillage farming systems is increasing the risk of infection in subsequent mungbean crops through the build-up of inoculum in soil. This increased risk of infection may be further exacerbated by retaining stubble infested with M. phaseolina on the soil surface, thereby increasing the amount of inoculum that could be splashed onto plant organs. Surface sterilisation, using sodium hypochlorite, significantly reduced colonisation levels in heavily colonised mungbean seed lines, but the process was not enhanced when a partial-vacuum was introduced to the process. In some seed lots, up to 32% of the seed was colonised only on the seed coat (defined here as contamination), whereas the remainder was internally infected. Surface sterilisation also reduced the overall colonisation of 132 commercial lines by approximately one-third. Storage of seed for one month at either 4°C or 15°C significantly reduced colonisation levels in seed, whereas freezing treatments did not. Eradication of M. phaseolina from mungbean seed was possible through thermotherapy. However, the conditions required for eradication also contributed to large increases in abnormal germination levels and large losses in overall germination - an unacceptable trade-off for sprouters. A combination of thermotherapy and surface sterilisation of colonised mungbean seed may provide a more efficient process of seed treatment.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.