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The intestinal microbiota is a massive and complex community, essential to the human host for good health and well-being. However, this population has been associated with gastrointestinal disease, and remains poorly understood. The aim of this study was to develop and validate DNA-based assays for the intestinal microbiota and to apply these methodologies to faecal samples collected from healthy volunteers and patients with gastrointestinal disease. Over 250 faecal samples were analysed using temporal temperature gradient gel electrophoresis (TTGE) and real time PCR. Validated assays had high sensitivity and reproducibility. Healthy individuals displayed a high level of temporal stability during short term studies (≤ 6 weeks) and long term studies (1-4years). Analysis of faecal samples provided by identical and fraternal twins demonstrated an influence of host genetics over the composition of the predominant bacteria in children. Two intervention studies, bowel lavage and the Atkins' diet, were carried out to monitor the impact of environmental change on the population's stability in healthy volunteers. Following bowel lavage, microbial populations rapidly recovered to control densities, however the stability of the population was disturbed. Introduction of the Atkins' diet, led to a significant change in the composition of the microbial population. A preliminary study of the intestinal microbiota in disease groups was undertaken. Significant differences were detected between inflammatory bowel disease groups and controls. Cluster analysis in these patients indicated a potential association between the composition of the predominant bacterial population and disease localisation. The studies reported here demonstrate that the faecal microbiota in healthy individuals is a highly stable population under the influence of both host genetics and environmental variables, however the population present in patients with inflammatory bowel disease exhibits differences compared to healthy controls.

The aims of this doctoral study were to: isolate naturally occurring bacteria, able to degrade microcystins (MCs), from New Zealand waterbodies; to understand the biological processes of microcystin degradation by bacteria; and to develop small scale biofilm technology for testing the effectiveness of bacteria for microcystin degradation and/or remediation. A significant amount of microcystins were required for biodegradation experiments. A modified method, using DEAE and Strata-X cartridge chromatography, was optimized for purifying microcystin variants from lyophilized bloom samples of the cyanobacterium Microcystis aeruginosa, collected en masse from Lake Horowhenua. Seven microcystin variants, MC-RR, MC-dMe-RR, MC-YR, MC-LR, [Dha7]MC-LR, MC-FR, and MC-AR were purified by chromatography and then identified by reverse-phase High Performance Liquid Chromatography (HPLC) with UV detector (UVD) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). A mixture of [Dha7]MC-LR and MC-LR, the main microcystin variants present, was used for examining biodegradation of microcystins by degrading bacteria. Three isolates of bacteria—NV-1, NV-2 and NV-3—purified from Lake Rotoiti, New Zealand were capable of degrading [Dha7]MC-LR and MC-LR. Among these isolates, NV-3 demonstrated the strongest degradative activity and was identified as a member of the genus Sphingomonas. On the basis of 16S rRNA sequencing, and 100% nucleotide sequence homology, it aligned most closely to strain MD-1. Based on the detection of two intermediate by-products (linearized peptides and a tetrapeptide) and the identification of four genes (mlrA, mlrB, mlrC and mlrD), that encode four putative proteins (enzymes) involved in microcystin degradation, it was suggested that the degradation of [Dha7]MC-LR and MC-LR by the Sphingomonas isolate NV-3 occurred by a similar mechanism previously described for Sphingomonas strain MJ-PV (ACM-3962). The bacterium Sphingomonas isolate NV-3 was examined for its ability to inhibit the growth of the cyanobacterium Microcystis aeruginosa strain SWCYNO4. It was found that the bacterium did not have any significant affect on the growth of the cyanobacterium, either by means of secretion of bacterial extracellular products or cell-to-cell contact between bacterial and cyanobacterial cells. It was established that Sphingomonas isolate NV-3 was a moderate biofilm former, based on two types of biofilm formation assays, namely, microtiter plate assays and coupon biofilm assays. This was carried out in preparation for using the bacterium in a bioreactor for biodegradation of [Dha7]MC-LR and MC-LR. The bacterium attached most effectively to ceramic, followed by PVC, polystyrene, stainless steel, and finally glass coupons. Biodegradation of MCs by the bacterium, in an internal airlift loop ceramic honeycomb support bioreactor (IAL-CHS bioreactor), was established in batch and continuous-flow experiments. In the batch experiment, NV-3 degraded a combination of [Dha7]MC-LR and MC-LR at an initial concentration of 25 µg/ml at 30 degrees C in 30 hours, whereas in the continuous-flow experiment, NV-3 degraded the same concentration of [Dha7]MC-LR and MC-LR in 36 hours with an hydraulic retention time (HRT) of 8 hours. This study has demonstrated that microcystin-degrading bacteria are present in New Zealand waterbodies and that these bacteria could be used, potentially on a larger scale, for removing microcystins from water.

The overall goal of this research was to determine what, if any, role grapevine rhizosphere bacteria play in the differing susceptibilities of New Zealand grown rootstocks to Cylindrocarpon black foot disease. The size and diversity of bacterial populations associated with the rhizospheres of grapevine rootstocks: 101-14, 5C, Schwarzmann and Riparia Gloire were evaluated. Dilution plating showed that total bacterial (P=0.012, P=0.005 for NA and KB, respectively) and fluorescent Pseudomonad (P=0.035) rhizosphere counts differed between rhizosphere and bulk soils but did not correlate with the differing susceptibilities of the rootstock varieties to black foot. No varietal differences were found for spore forming bacteria (P=0.201). SSCP banding patterns showed that species diversity was similar for most rootstocks, but that there were some differences in the composition of bacterial populations, probably attributable to vigour. Some functional characteristics of the bacteria isolated from the rhizospheres of the most and least susceptible rootstock varieties were assessed to investigate their potential to suppress the pathogen. In dual culture, bacteria from Riparia Gloire, 101-14 and the control soil all had little ability to antagonise Cylindrocarpon destructans. However, they differed in their degrees of activity for glucanase (P=0.000), protease (P=0.001) and siderophores (P=0.000). In all tests, bacterial isolates from the rhizosphere of 101-14 had the largest number of active isolates (P≤0.002); however, those from Riparia Gloire had the greatest degree of positive responses for the glucanase and siderophore assays. Bacterial isolates from the control soil produced few glucanases and no siderophores, but had the highest degree of protease activity. Bands excised and sequenced from SSCP gels frequently matched to other ‘uncultured bacteria’ in GenBank, as well as to other bacterial phyla, classes and genera commonly isolated from soil and sediment samples. These included members of the Firmicutes, Proteobacteria (α, δ, γ), Verrucomicrobia, Acidobacteria and Chromatiales. The pathogenicity of C. destructans and Fusarium oxysporum was investigated by inoculating soil containing wounded ungrafted rootstocks of 101-14, 5C, Schwarzmann and Riparia Gloire. Results indicated that F. oxysporum might be a more aggressive pathogen than C. destructans. Inoculation with F. oxysporum or C. destructans increased disease severity, P=0.018 and P=0.056, respectively at 0 cm. Rootstock variety influenced disease severity caused by C. destructans (P<0.001) and F. oxysporum (P=0.090), with rootstocks 101-14 and 5C being most susceptible to C. destructans, and Riparia Gloire and Schwarzmann most susceptible to F. oxysporum. There was also an indication that inoculation with one pathogen increased plant susceptibility to the other, with increased F. oxysporum infection in the C. destructans inoculated treatments of Riparia Gloire and Schwarzmann (P<0.05). The effect of carbohydrate stress (leaf trimming) and inoculation on C. destructans disease severity, incidence, and rootstock rhizosphere bacterial populations was evaluated by inoculating the soil containing one year old plants of Sauvignon Blanc scion wood grafted to rootstocks 101-14 and Schwarzmann. Disease severity and incidence was similar for both Schwarzmann (8.4% and 29.3%, respectively) and 101-14 (14.9% and 31.0%, respectively). When data for the moderate and no stress treatments were combined, because their effects were similar, the disease severity was significantly higher for the highly stressed plants(P=0.043). Stress did not influence disease incidence (P=0.551). Infection occurred in the non-inoculated plants, but disease severity was higher in the plants inoculated with C. destructans than those that were not. Root dry weight of highly stressed plants was lower than in both the moderately stressed (P=0.000) and unstressed plants (P=0.003). An interaction between inoculation and stress (P=0.031) showed that inoculated and highly stressed plants had the lowest root dry weight but there was no effect of rootstocks (P=0.062). There was no significant effect of carbohydrate stress (P=0.259) or inoculation (P=0.885) on shoot dry weight. SSCP banding patterns showed that bacterial diversity was generally similar between rootstocks, but stress and inoculation altered rhizosphere bacterial communities. This study has demonstrated that functionality of grapevine rhizosphere bacteria do differ between grapevine rootstock varieties that have different susceptibilities to black foot disease, but that this role needs to be further investigated if more accurate and practically relevant conclusions are to be drawn.

Early in 1962 the Forest Research Institute of the New Zealand Forest Service became aware that stands of second crop Pinus radiata (D. Don) on some areas of the Moutere Gravel formation were showing slow growth and had a chlorotic appearance (Fig. 1.1). The second crop followed clear felling of mature P. radiata trees and were aged from 0 to 15 years (stone and Will, 1965). It was felt that the apparent reduced growth of the second generation had much in common with similar productivity decline reported especially in European forestry literature. Stone and Will (1965) postulated that the immediate cause o£ the decline was a deficiency of nitrogen highlighted by the low levels of nitrogen in the leaves of the second crop trees, especially those growing on ridge sites. Numerous field trials have been laid out but many of the trials were poorly designed and consequently could not provide statistically sound results. However, some indication of nutrient deficiencies which occur on the Moutere Gravels were obtained. Even before these trials were laid down nutrient deficiencies had been highlighted by early attempts at farming. It was the partial failure of these crops that initially led to the planting of exotic pines, in the belief that these trees thrived on a limited supply of nutrients. The first crop of pines generally fulfilled expectations but nutrient deficiencies started to appear in extensive areas of the second crop. Accordingly, the Forest Research Institute made available three scholarships to study different aspects of the problem. Work was started on a study of the soil sequence across the Moutere Gravels to determine if there was a general decline in fertility of tho soil with the age of the soil and the environmental factors, such as climate which differs in the high inland areas and the low seaside areas of the Moutere Gravels, rather than a particular decline in fertility induced by the first crop of P. radiata. In another study the major weed species Ulex europaeus and Cytisus scoparius was studied to see if its value as a nitrogen fixer would outweigh its disadvantages as a silvicultural weed. Thirdly, a study of the microbial ecology of the soils was undertaken. Whyte (1966) reported that the second rotation trees started to increase their growth rate after approximately five years to a level paralleling the estimated growth rate of the first crop. It was postulated that the residues (needles, roots and branches) remaining after clear felling could cause an increase in microbial numbers and activity with a consequent immobilization of mineral nutrients which were not initially very plentiful. For this reason an area in Tasman Forest was selected in which mature trees and regeneration up to nine years old were found together to study microbial activity and numbers, energy dissipation and nitrogen dynamics to determine if immobilization of nutrients was causing the apparent declines.