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The effect of genetically modified crop plants on the microbiology of the rhizosphere was investigated using the single-gene Bt cotton as a case study. The project compared the rhizosphere microbiota of four Ingard® 1cotton plant varieties that were closely matched with their non-GM parental strains. The plants were grown in three different Australian soils, ie, a vertisol from a cotton-growing region, and two soils, a fine sandy loam and a red sand from South Australia that had not been exposed to cotton. At the time of the commencement of the project, the only commercially available genetically modified plants were cotton and carnations. The cotton industry in Australia is worth $1.5b annually, and care of the soil and the dynamics of its living microbial consortia needs to be understood for optimum management to enable agricultural sustainability. The general outline of the thesis incorporated four main sections: 1. Experimental setup and analysis of the soils and plants to be used, quantification of the Cry1A(c) plant-produced Bt protein, and its persistence in the soil environment. 2. Measurement of the selected microbial populations of bacteria, fungi, AMfungi, protozoans and nematodes, by counting and estimation by dilution and most-probable number methods. 3. Assessment of selected metabolic pathways to determine the effects on the soil microbial community by chemical and other biochemical methods 4. An overall analysis between different group ratios of expression of each of the variables tested, and the summary of the risk analysis and conclusion. The outcome of this work was the acquisition of scientific data to produce an environmental impact report. The findings of this study showed that generally the microbial populations and the products of major metabolic pathways correlated more closely within the non-GM and GM plant rhizospheres of the paired trials than those of separate trials, indicating that soil and plant cultivar had a stronger environmental effect. The results obtained from the paired trials did not show that there were consistent effects on the rhizosphere soil microbiota that could be attributed to the presence of the Cry1A(c) Bt plant protein on the selected strains of cotton plants. The results from the tests of the paired trials correlate highly with previously published work that the risk factors of genetically modified cotton plants on the microbiology of the rhizosphere soil were found be negligible and not consistent across trials. 1 ® Monsanto Co. St Louis, MO.

Copies of author's previously published articles in pocket. Bibliography: leaves 203-219. Main objectives of the thesis are: i. to verify a sealed chamber acid digestion technique for dissolution of plant material for total F analysis by a F ion selective electrode.--ii. to improve this technique for routine, rapid F analysis of plant material.--iii. to identify the inorganic ionic species of F which could be present in the soil solution.--iv. to determine which of these species are taken up by the plant root and those which are toxic to the plant.

Graduation date: 2003

In order to determine the impact of lesser snow goose (Chen caerulescens caerulescens) herbivory on above and belowground nutrient dynamics, a grazing and nutrient addition experiment was conducted in two habitat types utilized by snow geese near Churchill, Manitoba. Forage plant nutrition, soil microbial biomass, and inorganic and organic soil nutrients were measured in relation to the timing of grazing, over two consecutive summers. Soil was collected from the rhizosphere to determine the influence of foliar herbivory on root-microbe interactions. Primary productivity in both habitats is co-limited by the availability of both nitrogen (N) and phosphorus (P). Aboveground defoliation either caused a reduction or no change in soil microbial biomass nutrients (carbon (C), N, or P). Defoliated shoots had higher N concentrations and did not show compensatory growth within the season. Root biomass was somewhat reduced with grazing, but higher whole plant N content suggests that grazing does not compromise N-uptake.

In order to determine the impact of lesser snow goose (Chen caerulescens caerulescens) herbivory on above and belowground nutrient dynamics, a grazing and nutrient addition experiment was conducted in two habitat types utilized by snow geese near Churchill, Manitoba. Forage plant nutrition, soil microbial biomass, and inorganic and organic soil nutrients were measured in relation to the timing of grazing, over two consecutive summers. Soil was collected from the rhizosphere to determine the influence of foliar herbivory on root-microbe interactions. Primary productivity in both habitats is co-limited by the availability of both nitrogen (N) and phosphorus (P). Aboveground defoliation either caused a reduction or no change in soil microbial biomass nutrients (carbon (C), N, or P). Defoliated shoots had higher N concentrations and did not show compensatory growth within the season. Root biomass was somewhat reduced with grazing, but higher whole plant N content suggests that grazing does not compromise N-uptake.

Differential gene expression of P. aeruginosa in a rhizosphere biofilm on poplar tree roots was examined in order to identify new virulence factors from this human pathogen. Changes in gene expression for poplar trees contacted with P. aeruginosa was examined as well to identify the response of poplar roots to P. aeruginosa infection. This is the first study of the whole-transcriptome analysis of P. aeruginosa on a plant tree root. The 20 most highly-induced genes of P. aeruginosa were examined for their role in biofilm formation, rhizosphere colonization, barley germination, and poplar tree killing assays. Seven previously uncharacterized virulence genes (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150, and PA4295) were identified. The role of PA2663, a hypothetical protein discovered in the microarrays of P. aeruginosa while killing poplar trees, was examined in further detail. Expression of PA2663 protein increases biofilm formation in P. aeruginosa PAO1 drastically. By complementing the PA2663 mutation in trans and by studying with DNA microarrays and RT-PCR the PA2663 mutant vs. the wild-type strain, PA2663 was confirmed to be related to biofilm formation and was found that it is the first protein to control the psl operon in P. aeruginosa PAO1. Furthermore, PA2663 protein increases pyoverdine synthesis and quorum sensing (QS)- regulated phenotypes. A biofilm formation-related hypothetical protein, PA0939, was identified in this study. The effects of indole and 7-hydroxyindole on P. aeruginosa virulence factors were also examined for the first time. Indole and 7HI repressed expression of mexGHI-opmD multidrug efflux pump genes and genes involved in synthesis of QS-regulated virulence factors (pyocyanin, rhamnolipid, PQS, and pyoverdine production). In addition, the effects of an anti-cancer uracil analog, 5-fluorouracil (5-FU) on P. aeruginosa virulence factors and E. coli K-12 biofilm formation were examined. 5-FU repressed biofilm formation, abolished quorum-sensing phenotypes, and reduced virulence in P. aeruginosa. DNA microarray and biofilm studies with 5-FU in E. coli revealed that 5-FU controls biofilm formation through the AriR protein in E. coli K-12 strain. The effects of lsrR and lsrK mutations on E. coli biofilm formation were also examined by flow cell experiments.

In this study we attempt to use phytoremediation techniques to treat the contaminated soil of cadmium. The experiment is divided into two stages. In the first stage, we selected three different species of plants which could tolerate heavy-metals: vetiver (Vetiveria zizanioides), Pteris ensiformis cv. 'Victoriae' according to the past records, and Alternanthera philoxeroides (Mart.) Griseb, which were sampled from the metal contaminated site in Hunei, Kaohsiung county. These three species were planted in three pots with 10, 20 and 30 mg Cd kg-1 in soil respectively. After 9 weeks of the growth, the vetiver was found accumulating the highest Cd and grew better than the other two species. Therefore, we selected the species of vetiver in the second stage of experiment. First, the species of vetiver was planted in the pots with concentrations of 30 and 50 mg Cd kg-1 in soil respectively. Then the pots were put in the greenhouse for incubation. After the test was run for 210 days, we found that the species of vetiver was helpful in the increasing the number of species and amounts of each species of microbe ( total bacteria, fungi and actinomycete ), as well as dehydrogenase activity. Meanwhile, it was effective to decrease the bioavailability of cadmium. In addition, the infection rate of mycorrhizal fungi was increased , which showed that the species of vetiver could resist the cadmium stress in soils and stimulate the soil fertility. Finally, we use molecular biotechniques of PCR-DGGE to observe the microbial diversity in the contaminated soil. We found that the pots with 30 mg Cd kg-1 in soil had more number of bands than the pots with different Cd concentrations in soil, while the pots without vegetation was found more fruitful than vegetated pots. These experimental results indicated that the pots planted with the species of vetiver under this situation would help some special microorganisms to grow, and thus that the microbial diversity was reduced. The results also showed that the pots planted with vetiver with initial cadmium concentrations of 30 and 50 mg Cd kg-1 respectively, in soil exhibited the degradation rate of about 30 percent for both. It was not satisfied to this result in this study. However, the phytoextraction rates of cadmium were measured equal to 7.8 and 8.9 percent, respectively. According to these results, we suggested that the plant, which could hyperaccumulate heavy metals, might be used to increase the removable ability of cadmium in the future.

Individual isolates of the fungus Nectria haematococca exist in a wide range of habitats and part of this diversity is attributed to the presence of conditionally dispensable (CD) chromosomes that carry habitat-defining genes. In the current study a new factor located on one of these CD chromosomes was found. This trait allows pea pathogenic isolates of N. haematococca to grow in homoserine, a compound present in large amounts on pea root exudates. The gene(s) for homoserine utilization (HUT) are located on the same CD chromosome that carries the cluster of genes for pea pathogenicity, the PEP cluster. The PDA1 gene, a member of the PEP cluster, is routinely used as a marker for the presence of this CD chromosome, therefore it has been called the PDA1-CD chromosome. For the purpose of identifying the HUT gene(s), a physical map of the PDA1-CD chromosome was constructed. This map, in combination with synteny analysis, and Southern hybridizations led to the identification of a region of 365Kb that is likely to contain the HUT gene. By searching the publicly available genome of N. haematococca several candidates for HUT were identified.The synteny evaluation between the PDA1-CD chromosome and a different CD chromosome that carries the MAK1 gene, for chickpea pathogenicity, revealed a region (> 463Kb) of synteny, which advocates for a common ancestor for these CD chromosomes. However a large region (~ 1 Mb) in each of the CD chromosomes was found to carry unique DNA, therefore we proposed that individual isolates of this fungus contain large regions of unique DNA located on the CD chromosomes. The localization of syntenic regions also suggests that breakage points previous identified in the MAK1-CD chromosome could potentially be "hot spots" for recombination between both CD chromosomes. Furthermore, the anchoring of the PDA1-CD map to the genome of N. haematococca allowed the identification of additional putative habitat colonization genes present on both CD chromosomes, and niche-defining genes on the PDA1-CD chromosome.

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