Genotypic differences in growth and yield of two common bean (Phaseolus vulgaris L) cultivars to Rhizobium inoculation and management were investigated. In 2003-04, the two bean cultivars (Scylla and T-49) were combined with three inoculant treatments (strains CC 511 and RCR 3644, and a control of no inoculation), two fertiliser levels (0 and 150 kg N ha⁻¹) and two irrigation treatments (irrigated and rainfed). There was no nodulation on either cultivar. To further investigate the symbiotic relationship, 16 rhizobial isolates, including the two used in the first field experiment, were combined with the cultivar Scylla and evaluated in a greenhouse. Subsequently, five Rhizobium isolates were chosen for further field evaluation, based on signs of early nodulation in the greenhouse trial. The second field experiment in 2004-05 combined the five inoculant strains (RCR 3644, UK 2, H 20, PRF 81, PhP 17 and a control) with two bean cultivars (Scylla and T-49). In the greenhouse, nodule number varied from 7 (UK 2) to 347 (H 441) nodules plant⁻¹ at 51 DAS and from 13 (UK 1) to 335 (CIAT 899) nodules plant⁻¹ at 85 DAS. In 2004-05, in the field, nodulation was also variable, ranging between 1 and approximately 70 nodules plant⁻¹, with higher nodules numbers plant⁻¹ being found on cultivar T-49. Of the isolates used in the field, strains H 20, PRF 81 and PhP 17 produced 70, 25 and 12 nodules plant⁻¹ at 70, 40 and 54 DAS respectively. Nodules formed were of various sizes and more than 80 % were pink to dark red in colour denoting the presence of leghaemoglobin and active N fixation. The remaining nodules were either green or white. The importance of selecting an appropriate cultivar for the growing conditions was highlighted in these experiments. Leaf area index, leaf area duration intercepted radiation and final utilisation efficiency were significantly affected by cultivar. In both seasons cv. T-49 reached maturity (dry seed) before Scylla, while unirrigated plants reached green pod maturity seven days before irrigated plants. Plants of cv. Scylla gave a final TDM of 730 g m⁻²; compared to the 530 g m⁻² produced by T-49. The average growth rate was 7.0 and 5.2 g m⁻² day⁻¹ for Scylla and T-49 respectively (2003-04). Plants receiving 150 kg N ha⁻¹ produced 665 g m⁻² TDM which was 12 % more than was produced by unfertilised plants. The application of 150 kg N ha⁻¹ gave an average growth rate of 6.4 g m⁻² day⁻¹ compared to 5.7 g m⁻² day⁻¹ from plants with no N. Inoculation in the field had no significant effect on TDM in both seasons. Temperature affected growth and DM accumulation. Accumulated DM was highly dependent on cumulative intercepted PAR. Air temperatures below the base temperature (10 °C) affected growth in 2004-05, resulting in plants accumulating just 0.24 g DM MJ⁻¹ PAR during early growth. This increased to 2.26 g DM MJ⁻¹ PAR when the temperature was increased above the base temperature. There was a strong relationship between LAI and intercepted PAR. A LAI of 4.0-4.5 was required to intercept 90-95 % of incident solar radiation. Cultivar significantly (p < 0.001) affected radiation use efficiency (RUE). Scylla had a RUE of 1.02 g DM MJ⁻¹ PAR compared to T-49 at 1.18 g DM MJ⁻¹ PAR. Seed yield was significantly (p < 0.001) affected by cultivar and fertiliser application. Cultivar Scylla produced 467 g m⁻² which was 76 % more than T-49, while a 12 % increase in seed yield was observed in N fertilised plants over unfertilised plants. Only cultivar significantly affected HI, while the yield components that had the greatest effect on seed yield were hundred seed weight and pods plant⁻¹. Inoculation significantly (p< 0.05) affected 100 seed weight (2004-05). Plants inoculated with strain H 20 had the highest 100 seed weight at 25.2 g with cv. Scylla producing larger seeds than T-49. The belief that local environmental conditions play a major role on field survival of bacteria, led to the use of PCR methods to identify field nodulating organisms. Amplification of genomic DNA from parent isolates using primers fC and rD generated a single band for each isolate. Isolates were identified to the species level as either Rhizobium or Agrobacterium, using the highly conserved internally transcribed spacer (ITS) region and are known to nodulate common bean. The DNA extracted from the isolates recovered from nodules of field grown beans gave multiple bands with primers fC and rD. Five distinct banding patterns were observed. All of these were different from those of parent isolates. Sequencing of the 16S rRNA demonstrated that nodules of field grown beans in Canterbury were inhabited by Pseudomonads either alone or in association with other root nodulating organisms. The inability to identify the inoculant strains in nodules of field grown beans does not rule out their infection and nodulating function in the cultivars used. The results suggest the possibility of both Rhizobium and Pseudomonads cohabiting in the nodules of field grown beans. The aggressive nature of Pseudomonads on artificial media, possibly out competing the inoculant rhizobia is proposed, leading to the inability to identify the inoculant strain from the nodules of the field grown beans by PCR methods. The need to identify the nodule forming or nodule inhabiting bacteria in the nodules is necessary to classify the importance of these organisms and their economic benefit to agricultural production. This study also underlines the importance of using PCR methods to gain valuable insights into the ecological behaviour of Rhizobium inoculants and nodule inhabiting organisms.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 µm²) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.

A series of experiments was carried out on cultivated oat (Avena sativa L. cv Amuri) to examine the efficacies of fluazifop-butyl and glyphosate against water stressed plants, plants grown in low and high nitrogen and plants treated with gibberellic acid (GA₃). Avena sativa L. was used as a test plant and on completion of the experiments, further studies were carried out on the weed species wild oat (Avena fatua L.). In the laboratory, plants maintained at wilting point for five days before and nine days after spraying and treated with fluazifop-butyl (0.5 kg a.i./ha) appeared healthy 32 days after herbicide application, while plants supplied with water throughout the experiment were completely chlorotic/necrotic and had main stem detachment from within the leaf sheaths. In the field, plants maintained unirrigated until 14 days after spraying with fluazifop-butyl (0.25 kg a.i./ha) or glyphosate (0.18 kg a.i./ha) showed greater tolerance to the herbicides than plants irrigated regularly. Values for seed head yield for water stressed and irrigated plants, 45 days after applying fluazifop-butyl, were 66 g and zero g dryweight/m² respectively. Comparable values for glyphosate treated plants were 65 g and 25 g dryweight/m². Radiolabel studies indicated that in comparision with well watered plants, water stressed plants absorbed 20% less applied ¹⁴C-glyphosate. In addition, the proportion of absorbed ¹⁴C-glyphosate translocated from the treated leaf was 15% less under water stress conditions. Uptake of ¹⁴C-fluazifop-butyl was similar under well watered and water stress conditions and was 30-40% of that applied. The proportion of absorbed ¹⁴C-activity which was transported was very low, but was greater under well watered conditions (7.6%) than under water stress conditions (4.4%). Under well watered conditions in the laboratory and field, fluazifop-butyl (0.25 kg a.i./ha) and glyphosate (0.18 kg a.i./ha) were less toxic at low nitrogen than high nitrogen. For example, 34 days after spraying with fluazifop-butyl under laboratory conditions total plant dry weight was 1.51 g and 0.56 g at 1.0 mol/m³ and 10 mol/m³ applied nitrate respectively. As with soil water content, soil nitrogen content had no effect on uptake of fluazifop-butyl. However, the proportion of absorbed fluazifop-butyl which was translocated out of the treated lamina was greater under high nitrogen conditions (26.1 %) than under low nitrogen conditions (9.3%). Under laboratory conditions, addition of 200 µg GA₃to the leaf sheaths two days prior to spraying with fluazifop-butyl or glyphosate increased the efficacy of both herbicides at low nitrogen. Similarly, under field conditions application of GA₃ (0.21 kg/ha) two days prior to spraying with glyphosate increased the performance of the herbicide against Avena sativa L. growing in a nitrogen depleted soil. At harvest, seed head yield for GA₃ treated and non-treated plants was zero and 7.4 g dry weight/m² respectively. Experiments with Avena latua L. showed that this species was tolerant of fluazifop-butyl and glyphosate when grown in low water or low nitrogen conditions. Under water stress conditions, pre-treatment with GA₃ increased the phytotoxicity of fluazifop-butyl to Avena latua L. Similarily, GA₃ enhanced the phytotoxicity of glyphosate to Avena latua L. grown under low nitrogen conditions. Reduced performance of fluazifop-butyl under stress conditions involves a reduction in translocation of herbicide to meristems, but other factors are likely to be involved. It was concluded that for glyphosate, reductions in uptake and translocation of the herbicide are important factors causing reduced performance of this herbicide under stress conditions. Possible reasons for GA₃ enhancement of fluazifop-butyl and glyphosate activity under stress conditions are discussed and the potential of growth regulators as adjuvants is considered.

The vegetative and reproductive development of balansa clover (Trifolium michelianum Savi.) were quantified in relation to the environmental drivers of each phenophase in field and controlled environments. In a grazed experiment over 6 years, balansa clover sown with cocksfoot (Dactylis glomerata) contributed 1.6 t DM/ha/year, or ~20% of the total DM production. However, grazing management for increased seed production during flowering in the establishment year strongly influenced balansa clover regeneration. The earliest closed plot (September) averaged between 2.2 and 4.3 t DM/ha/year of balansa clover across all six years. In an incubator, balansa clover required 29°Cd for germination with an optimum temperature of 14°C and a maximum of 40°C. The base temperature for germination was 0°C. A field experiment determined that 38°Cd were required for emergence with an optimum soil temperature (Topt) of 8.5°C. The time from emergence until the first leaf appeared, the phyllochron and timing of axillary leaf appearance were compared with perennial ryegrass (Lolium perenne) and white clover (Trifolium repens L.). The rate of each was found to increase linearly with temperature. The balansa clover cultivar ‘Frontier’ required 97°Cd from sowing for the first leaf to appear, had a phyllochron of 47°Cd and secondary leaves appeared after 490°Cd. For each vegetative stage, the base temperature was 2.5°C. The timing of flower appearance depended on the quantity and direction of change of the photoperiod at emergence. A balansa clover plant, cv. ‘Bolta’, which emerged on 1 December into an increasing photoperiod of 15.6 hours flowered after 574°Cd (Tbase = 2.5°Cd) or 58 days after emergence. In contrast, if the plant emerged on 16 January into a similar but decreasing photoperiod it took 1503°Cd or 227 days to flower. This length of time became progressively shorter until remaining constant after the shortest day. In contrast, ‘Frontier’ took a constant 390 and 690 °Cd in increasing and decreasing photoperiods, respectively. The time which an individual inflorescence took from pollination until seeds were physiologically mature was 250 °Cd for both ‘Bolta’ and ‘Frontier’. The re-establishment of balansa clover each year relied on a large seed set (>1000 kg/ha) in the establishment year. The continued survival of balansa clover would then depend on a similar seeding event within a 4-5 year period to maintain the seed bank. Management considerations for balansa clover persistence and survival are discussed.

In this thesis, experiments were conducted in the laboratory and the field to determine whether the provision of floral resources to hoverflies could enhance the biological control of aphids. The overall aim was to clarify hoverfly behaviour and ecology in an agroecosystem in order to understand the potential of these insects for biocontrol under a conservation biological control (CBC) regime. A preliminary experiment in New Zealand compared the effect of different coloured water-traps on catches of the hoverflies Melanostoma fasciatum (Macquart) and Melangyna novaezelandiae (Macquart). Significantly more individuals were caught in completely yellow traps than in traps with green outer walls and yellow inner walls or in completely green traps. This suggested that if a measure of hoverfly numbers relating to a particular distance along a transect is required, consideration should be given to the ability of hoverflies to detect yellow traps from a distance. The use of traps that are green outside would more accurately reflect the local abundance of hoverflies, as the insect would be likely to see the yellow stimulus only when above or close to the trap. Also, the addition of rose water significantly increased the number of M. fasciatum caught. From a suite of flowering plants chosen for their ability in other studies to increase hoverfly visit frequencies, laboratory experiments were conducted in France to determine the plant’s effectiveness at enhancing Episyrphus balteatus (De Geer) ‘fitness’, and to evaluate whether adult feeding on flowers was related to performance. Phacelia (Phacelia tanacetifolia Bentham cv. Balo), followed by buckwheat (Fagopyrum esculentum Moench cv. Katowase) and coriander (Coriandrum sativum L.) gave the optimal reproductive potential of female E. balteatus. There was no correlation between pollen and nectar consumption, and there was no discernible positive correlation between the quantity of pollen ingested and the resulting female performance. Phacelia and buckwheat were then studied as resource subsidies in the field in New Zealand. The effect of incorporating phacelia or buckwheat in the margins of 5 m x 5 m broccoli plots was tested for hoverfly activity and floral ‘preferences’. Hoverflies which had fed on phacelia and buckwheat pollen were found up to 17.5 m from the floral strips and females of M. fasciatum and M. novaezelandiae consumed more phacelia pollen than that of buckwheat in the field. These results support the choice of phacelia as an ideal floral resource subsidy in crops for enhanced biological control by these New Zealand species. The need for studying hoverfly movement in a large-scale field experiment was apparent from the field studies, so the next experiment was carried out in a field 450 × 270 m and flies were marked via their ingestion of the pollen of phacelia. The focus was on the proportion of flies having consumed the pollen. Although large quantities of pollen were found in some hoverfly guts, most did not contain phacelia pollen and very few were captured at 50 m from phacelia, compared with numbers at the border of the floral strip. A possible explanation was that hoverflies feed on a large variety of pollen species, reducing the relative attraction of phacelia flowers. Another possibility was that hoverflies dispersed from the phacelia away from the crop. Also, pollen digestion rates are likely to be a factor. Finally, a series of experiments was conducted in the field and laboratory to study hoverfly efficacy through oviposition and larval behaviour. In field experiments, female M. fasciatum and M. novaezelandiae laid more eggs where buckwheat patches were larger; however higher oviposition rates did not lead to improved aphid population suppression. In greenhouse experiments, larvae of E. balteatus could initiate a decline in aphid numbers at the predator: prey ratio 1: 8.3, however this control did not persist. Experiments in the laboratory showed that hoverfly larvae became more active and left the system while aphid numbers declined or numbers of larvae increased. This behaviour was caused by two factors: hunger and avoidance of conspecific larvae. Further experiments showed that the avoidance of conspecifics was caused by mutual interference rather than cannibalism. The results of this work highlight the importance of hoverfly dispersal ability. Given the observations of foraging behaviour of females and mutual interference observed between larvae, and the lack of success in CBC by hoverflies in experiments at the crop scale, it is essential to assess the impact of insect predators and parasitoids at a landscape scale.

The Tasmanian lacewing (Micromus tasmaniae Walker) is one of the most common aphid predators occurring in lucerne crops in New Zealand. A comparison of sampling techniques, and the output from a simulation model, suggest that the abundance of this lacewing may have been significantly underestimated in the past. Although the occurrence of aphid predators was erratic M. tasmaniae occurred more often and in far greater numbers (up to 100 m⁻²) than any other predator species. A simulation model for lacewing development in the field indicated that the large adult populations which occurred could be accounted for on the basis of reproductive recruitment. Independent evidence that immigration was not involved in the occurrence of these large populations was gathered using directional flight traps around the field perimeter. The major factors influencing lacewing population dynamics were the availability of aphid prey and, in the autumn, parasitism. Otherwise, survival of all life-histoty stages was high with no evidence of egg or larval cannibalism. Several instances of high lacewing mortality were identified by the model and the lack of any obvious cause for these highlights inadequacies in the understanding of lacewing bionomics. The model, which used a linear relationship (day-degrees) between development and temperature, was incapable of accurately predicting lacewing emergence under field temperatures which fluctuated outside the linear region of the development rate curve. Temperature thresholds and thermal requirements estimated under fluctuating temperatures similar to those in the field produced almost identical model output to those estimated under constant temperatures in the laboratory. Prey species was capable of influencing the rate of lacewing development. M. tasmaniae has the attributes necessary to produce large populations in the short time available between lucerne harvests. The asymptote of the functional response curve is low but the efficiency at converting aphids to eggs is high. Therefore, the lacewing is able to attain maximun reproductive output at low prey densities. A low temperature threshold for development (4-5° C), rapid development and short preoviposition period results in a short generation time (49 days at 15° C). Long adult life, high fecundity and the absence of any form of estivation or diapause, results in complete overlap of generations and multiple generations per year. M. tasmaniae's role as an aphid predator is restricted by its low appetite for prey and by the lucerne management regime currently practiced in New Zealand. Because it consumes relatively few aphids per day the lacewing's ability to destroy large aphid populations is limited. However, this may be offset by its ability to attack aphids early in the aphid population growth phase, and by the large numbers of lacewings which may occur. Under the present lucerne management schemes the large lacewing populations which do occur are forced out of the fields, or die, following harvest. A number of management options for increasing the lacewings impact as an aphid predator are briefly discussed.

Despite the importance of a high white clover (Trifolium repens) content in temperate pastoral systems in terms of livestock performance and nitrogen fixation, the proportion of white clover in grass-clover pastures is often low (<20%). This thesis examined in two experiments whether the white clover content of pastures could be improved by sowing white clover with alternative grass species to diploid perennial ryegrass (Lolium perenne L.). In a pasture experiment, DM production, pasture composition and morphology of grass-clover mixtures was measured over the establishment year (January 2007 to January 2008) where white clover was sown in fine mixtures with diploid perennial ryegrass, tetraploid perennial ryegrass, timothy (Phleum pratense L.) and cocksfoot (Dactylis glomerata L.). Pastures were irrigated and rotationally grazed with on-off grazing with Coopworth ewe hoggets. Total annual DM production of pasture was more than 20% higher in tetraploid (12521 kg DM ha⁻¹) and diploid (11733 kg DM ha⁻¹) perennial ryegrass than timothy (9751 kg DM ha⁻¹) and cocksfoot (9654 kg DM ha⁻¹). However, timothy (5936 kg DM ha⁻¹) and cocksfoot (5311 kg DM ha⁻¹) had more than four times higher white clover annual DM production than tetraploid (1310 kg DM ha⁻¹) and diploid (818 kg DM ha⁻¹) ryegrass. Pasture growth rate at the first three harvests in autumn was significantly greater in tetraploid and diploid ryegrass than timothy and cocksfoot. Timothy and cocksfoot had a higher proportion of white clover than tetraploid and diploid perennial ryegrass throughout the entire year. This was due to more and larger white clover plants in timothy and cocksfoot plots. In a grazing preference experiment, the partial preference of sheep for white clover offered in combination with the same grass species as in the pasture experiment was measured in five grazing tests in May, September, October, November and December 2007. Pastures were sown in January 2007. Paired plots (grass and clover both 4.2 m x 10 m) were grazed by three Coopworth ewe hoggets between 9am and 5pm, and preference was recorded by decline in pasture mass and visual scan sampling for grazing time. Grazing preference for clover was generally low throughout these tests (e.g. average apparent DM intake from clover = 47%; average grazing time from clover = 44%). Several explanations are proposed for this low preference including a high N content and intake rate of the grass relative to the clover. No significant differences were found among the grass treatments in total grass grazing time, total clover grazing time, ruminating time, the proportion of grazing time on clover, selective coefficient for clover and DM intake percentage from clover at any date. There was no significant change in overall sward surface height (SSH) decline among grass treatments throughout all the tests except December 2007 when the overall SSH decline for cocksfoot was significantly lower than the other species. The study indicated that the rapid growth rate of perennial ryegrass in the early phase of pasture establishment, rather than differences in partial preference, was the key factor limiting white clover content in the mixed swards relative to cocksfoot and timothy pastures. It is concluded that high clover-containing pastures capable of delivering high per head performance can be established through the use of slow establishing pasture species such as timothy and cocksfoot.

Take-all, caused by the soilborne fungus, Gaeumannomyces graminis var. tritici (Ggt), is an important root disease of wheat that can be reduced by take-all decline (TAD) in successive wheat crops, due to general and/or specific suppression. A study of 112 New Zealand wheat soils in 2003 had shown that Ggt DNA concentrations (analysed using real-time PCR) increased with successive years of wheat crops (1-3 y) and generally reflected take-all severity in subsequent crops. However, some wheat soils with high Ggt DNA concentrations had low take-all, suggesting presence of TAD. This study investigated 26 such soils for presence of TAD and possible suppressive mechanisms, and characterised the microorganisms from wheat roots and rhizosphere using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A preliminary pot trial of 29 soils (including three from ryegrass fields) amended with 12.5% w/w Ggt inoculum, screened their suppressiveness against take-all in a growth chamber. Results indicated that the inoculum level was too high to detect the differences between soils and that the environmental conditions used were unsuitable. Comparison between the Ggt DNA concentrations of the same soils collected in 2003 and in 2004 (collected for the pot trial), showed that most soils cropped with 2, 3 and 4 y of successive wheat had reduced Ggt DNA concentrations (by 195-2911 pg g-1 soil), and their disease incidences revealed 11 of the 29 test soils with potential take-all suppressiveness. Further pot trials improved the protocols, such that they were able to differentiate the magnitudes of suppressiveness among the soils. The first of the subsequent trials, using 4% w/w Ggt inoculum level, controlled conditions at 16°C, 80% RH with alternate 12 h light/dark conditions, and watering the plants twice weekly to field capacity (FC), screened 13 soils for their suppressiveness against take-all. The 13 soils consisted of 11 from the preliminary trial, one wheat soil that had been cropped with 9 y of wheat (considered likely to be suppressive), and a conducive ryegrass soil. The results revealed that 10 of these soils were suppressive to take-all. However, in only four of them were the effects related to high levels of microbial/biological involvement in the suppression, which were assessed in an experiment that first sterilised the soils. In a repeat trial using five of the soils H1, H3, M2, P7 (previously cropped with 3, 3, 4 and 9 y successive wheat, respectively) and H15 (previously cropped with 5 y of ryegrass), three of them (H1, H3 and M2) had reduced Ggt DNA concentrations (>1000 pg g-1 soil reductions), and were confirmed to be suppressive to take-all. A pot trial, in which 1% of each soil was transferred into a γ-irradiated base soil amended with 0.1% Ggt inoculum, indicated that soils H1 and H3 (3 y wheat) were specific in their suppressiveness, and M2 (4 y wheat) was general in its suppressiveness. The microbial communities within the rhizosphere and roots of plants grown in the soils, which demonstrated conduciveness, specific or general suppressiveness to take-all, were characterised using PCR-DGGE, and identities of the distinguishing microorganisms (which differentiated the soils) identified by sequence analysis. Results showed similar clusters of microorganisms associated with conducive and suppressive soils, both for specific and general suppression. Further excision, re-amplification, cloning and sequencing of the distinguishing bands showed that some actinomycetes (Streptomyces bingchengensis, Terrabacter sp. and Nocardioides sp.), ascomycetes (Fusarium lateritium and Microdochium bolleyi) and an unidentified fungus, were associated with the suppressive soils (specific and general). Others, such as the proteobacteria (Pseudomonas putida and P. fluorescens), an actinomycete (Nocardioides oleivorans), ascomycete (Gibberella zeae), and basidiomycete (Penicillium allii), were unique in the specific suppressiveness. This indicated commonality of some microorganisms in the take-all suppressive soils, with a selected distinguishing group responsible for specific suppressiveness. General suppressiveness was considered to be due to no specific microorganisms, as seen in soil M2. An attempt to induce TAD by growing successive wheat crops in pots of Ggt-infested soils was unsuccessful with no TAD effects shown, possibly due to variable Ggt DNA concentrations in the soils and addition of nutrients during the experiment. Increasing numbers of Pseudomonas fluorescens CFU in the rhizosphere of plants, during successive wheat crops was independent of the Ggt DNA concentrations and disease incidence, suggesting that increases in P. fluorescens numbers were associated with wheat monoculture. This study has demonstrated that TAD in New Zealand was due to both specific and general suppressiveness, and has identified the distinguishing microorganisms associated with the suppression. Since most of these distinguishing microorganisms are known to show antagonistic activities against Ggt or other soilborne pathogens, they are likely to act as antagonists of Ggt in the field. Future work should focus on validating their effects either individually, or interactively, on Ggt in plate and pot assays and under field conditions.

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

This study consisted of two sheep grazed dryland pasture experiments. Experiment l compared sheep production from 3-year-old cocksfoot based pastures grown in combination with white, Caucasian, subterranean or balansa clover with a ryegrass-white clover pasture and a pure lucerne forage. Sheep liveweight gain per head from each pasture treatment and the pure lucerne stand was recorded in the 2006/07 and 2007/08 seasons. The cocksfoot-subterranean clover pasture provided equal (381 kg LW/ha in 2006) or higher (476 kg LW/ha in 2007) animal production in spring and gave the highest total animal production (646 kg LW/ha) averaged across years of the five grass based pastures. However, total annual liveweight production from lucerne was higher than any grass based pasture mainly due to superior animal production during summer when lucerne provided 42-85% higher animal production than any of the grass based pastures. In Experiment 2, the effect of stocking rate (8.3 (low) and 13.9 (high) ewes + twin lambs/ha) and time of closing in spring on lamb liveweight gain, pasture production and subterranean clover seedling populations was monitored over 2 years for a dryland cocksfoot-subterranean clover and ryegrass-subterranean clover pasture in Canterbury. In both years, twin lambs grew faster (g/head/d) in spring at low (327; 385) than high (253; 285) stocking rate but total liveweight gain/ha (kg/ha/d) was greater at high (7.26; 7.91) than low (5.43; 6.38) stocking rate. Ewes also gained 0.5 and 1.5 kg/head at the low stocking rate in 2006 and 2007 respectively but lost 0.2 kg/head in 2006 and gained 0.3 kg/head at high stocking rate in 2007. Mean subterranean clover seedling populations (per m²) measured in autumn after grazing treatments in the first spring were similar at both low (2850) and high (2500) stocking rate but declined with later closing dates in spring (3850, 2950, 2100 and 1700 at 2, 4, 6, 8 weeks after first visible flower). Seedling populations measured in autumn after grazing treatments in the second spring were also unaffected by stocking rate (low 1290, high 1190) but declined with later closing dates in spring (1470, 1320 and 940 at 3, 5 and 8 weeks after first flowering, respectively). The effect of stocking rate and closing dates in spring on pasture and clover production in the following autumn was similar to the effects on seedling numbers in both years. However, clover production in the following spring was unaffected by stocking rate or closing date in the previous year at the relatively high seedling populations generated by the treatments. This was presumably due to runner growth compensating for lower plant populations in pastures that were closed later in spring. Subterranean clover runner growth in spring may not compensate in a similar manner if seedling numbers in autumn fall below 500/m². Mean annual dry matter production from cocksfoot and ryegrass pastures grown with and without annual clovers pasture production ranged from 6.4 to 12.4 t DM/ha/y but stocking rate (8.3 vs. 13.9 ewes/ha) during spring did not affect annual pasture production. Pastures overdrilled with annual clovers yielded 23-45% more dry matter production than pastures grown without annual clovers. The study confirms the important role of subterranean clover in improving pasture production and liveweight gains of sheep in dryland cocksfoot and ryegrass pastures. Lowering stocking rate from 13.9 to 8.3 ewes/ha was a less effective method of increasing seed production of subterranean clover in dryland pastures although it did lead to increased liveweight gain per head.