Thesis (Ph.D.)--University of Delaware, 2005. / Principal faculty advisor: Anne S. Robinson, Dept. of Chemical Engineering. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin -- La Crosse, 2006. / Includes bibliographical references (leaves 69-73)

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). Two offprints bound in the back. Includes bibliographical references (leaves 142-162). Also available via the World Wide Web.

Crop rotation is a key strategy of sustainable agriculture in the Canadian Prairie. Improving crop productivity and yield stability in pulses-based cropping systems with better soil biology is the ultimate goal of this research. Firstly, my studies provide information on the effect of pulses on the biodiversity of soil fungi: arbuscular mycorrhizal fungi (AM) and non-AM fungi, associated with the main pulse species grown in the Canadian Prairie (field pea, lentil, and chickpea), and their influence on wheat-based cropping systems. Secondly, the optimum 4-yr crop rotation for wheat production was determined, based on the relationship among fungal communities associated to the different crops and the yield and quality of these crops. My research included two experiments. First, in a field experiment replicated in time and site, the effect of previous pulse crops on wheat root-associated microbial communities and crop performance was assessed in four 2-yr rotation systems. Second, a 4-yr field experiment evaluated the relative influence of eight different crop rotations on root-associated microbial communities and on wheat productivity in the last year of the rotations. A greenhouse assay was conducted to evaluate, under controlled conditions, the influence of the microbial communities selected by these previous field crop rotations on wheat performance, using soil from the field as inoculant. The response of root-associated microbial communities was characterized using next generation sequencing technologies, phospholipid fatty acid markers, microscopic observation of roots and soil dehydrogenase assay. Plant response was evaluated based on crop density, biomass, yield and tissue nutrient content. My studies showed that community composition of AM and non-AM fungal communities in the roots of wheat were largely influenced by host plant identity and environmental conditions. The structure of the overall fungal community in wheat roots was not affected by the previous crops. The soil microbial legacies of previous crops were different from the fungal communities found in the roots of the following wheat, suggesting that wheat, as a host plant, selects and associates with a specific fungal community. Seasonal variations in soil moisture, temperature, pH, and nutrient cycling between sampling times have a great influence on soil microbes and could also be influencing these effects. The 2-yr crop rotation experiment revealed that wheat after a pulse crop had higher plant density and produced more seed biomass and total yield. The 4-yr crop rotation studies revealed that, in the field, diversified rotations including pea or lentil in alternate years, largely contributed to wheat performance. However, rotations including chickpea contributed little to the rotation benefits, suggesting that a careful selection of plant species is essential to improve the performance of the agroecosystem. Contrary to the field results’ findings, under greenhouse conditions, rotations that included chickpea before wheat contributed the best to wheat productivity, suggesting that in the field, factors other than the microbial community selected by chickpea were responsible for the poor performance of chickpea-wheat rotations in the field. Soil bacterial and fungal biomasses were positively correlated with wheat yield in the field experiments, which suggests that an abundant and diversified microbial community positively influences wheat productivity. Also, possible antagonistic and synergistic interactions between different AM species and root pathogens could be inferred. These results suggest that many AM fungi can potentially contribute to combat pathogens and enhance plant performance, whereas other might produce detrimental effects on the plants. Overall my studies revealed that host plant identity and environmental conditions influence the fungal community structure and dynamics. The frequency and sequence of crops in the rotations strongly influences productivity in wheat based agroecosystems. Lentil and pea alternating with wheat largely contribute to wheat performance. Thus, the productivity of wheat can be improved by selecting and including the plant species most beneficial to the rotation in order to increase soil available water and N, while promoting beneficial microbial associations and reducing disease incidence.

Resumo: Extratos etanólicos do ritidoma, da casca do caule e do fruto e da semente de Swartzia argentea, S. laevicarpa, S. panacoco, S. polyphylla e de S. sericea foram testados para os fungos degradadores de madeira. O volume de 10 mL de cada extrato nas concentrações de 0, 1; 0,01 e 0,001 mg/ L foi adicionado a 90 mL de meio de cultura agar-malte e inoculado com Pycnoporus sanguineus (L.:Fr.) Murr., Trametes villosa (Fr.) Ryv. e Lenzites trabea Pers.:Fr. A área (cm2) da colônia do fungo e o índice antifúngico (IAF) dos extratos foram calculados. A diferença significativa entre as médias do crescimento dos fungos (cm2) foi observada nos extratos do ritidoma, da casca do fruto e da semente, independente da espécie de Swartzia, mas para os extratos da casca do caule não houve diferença significativa para S. laevicarpa e para S. sericea. Houve variação significativa entre as médias da área micelial (cm2) dos fungos para cada concentração do extrato. Os extratos dos ritidomas de S. argentea, S. polyphylla e de S. sericea e da casca do caule de S. panacoco apresentaram o índice antifúngico de 100% para as três espécies de fungos. Estes extratos foram submetidos à partição líquido-líquido com solventes de polaridade crescente, obtendo-se as frações: hexânica, diclorometano, acetato de etila e aquosa, visando a atividade antifúngica das frações em ensaio de bioautografia. As frações: diclorometano, acetato de etila e aquosa, foram aplicadas em concentrações diferentes para cada espécie de Swartzia em cromatofolha de gel de sílica GF254, sendo que a fração aquosa foi eluída com a fase móvel butanol:etanol:agua (4,0;1,1;1,9), a fração acetato de etila com o sistema de solvente diclorometano:acetato de etila:metanol (5:3:2), acrescido de 0,01 mL de ácido acético, e a fração diclorometano com os sistema de solventes diclorometano:acetato de etila (7:3)...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Ethanolic extracts from the ritidome, the barks of the stem and of the fruit, and the seed of Swartzia argentea, S. laevicarpa, S. panacoco, S. polyphylla and S. sericea were tested for decay fungi. Pycnoporus sanguineus (L.:Fr.) Murr., Trametes villosa (Fr.) Ryv. and Lenzites trabea Pers.:Fr. were inoculated in 90 mL of Malt Agar plus 10 mL of each extract, in the concentrations of 0,1, 0.01 and 0,001 mg/mL. Colony area (cm2) and antifungal index of the extracts were calculated. Significant difference among the fungal growth average (cm2) was observed in the extracts of the ritidome, the barks of the fruit and of the seed, despite the species studied. However, there was no difference in the extracts of the barks of the stem in S. laevicarpa and S. sericea. There was significant variation among the averages of the fungal mycelial area (cm2) in each extract concentration. The extract of the ritidome of S. argentea, S. polyphylla, and S. sericea, besides one from the barks of the stem of S. panacoco, resulted in 100% antifungal index for all fungal species. These extracts were submitted to liquid-liquid partition with solvents of increasing polarity resulting the following fractions: hexanic, dichloromethane, ethyl acetate and water, aiming the fraction antifungal activity in thin layer cromatography TLC. Dichloromethane, ethyl acetate and the watery fraction were applied in different concentrations on each Swartzia species studied, in TLC GF254. The watery fraction was eluted with butanol:ethanol:water (4,0; 1,1; 1,9), the fraction ethyl acetate with the solvent system dichloromethane:ethyl acetate: methanol (5:3:2) plus 0,01mL acetic acid, and the dichloromethane fraction with the solvent system dichloromethane: ethyl acetate (7:3). The chromatograms were inoculated with P. sanguineus, T. villosa and L. trabea in Malt Agar, and chromatographic strips with no fractions...(Complete abstract click electronic access below) / Orientador: Vera Lucia Ramos Bononi / Coorientador: Ana Cláudia Fernandes Amaral / Banca: Mauricio Bacci Junior / Banca: Dejanira de Franceschi de Angelis / Banca: Luiz Carlos Couto / Banca: Mario Tomazello / Doutor

Protein N-glycosylation, which is ubiquitous in eukaryotes, is a complex pathway involving numerous gene families. Early stages of the glycosylation pathway show a high degree of conservation among eukaryotes, yet diversification of the number and size of gene families involved in the later stages of the pathway has led to the evolution of increasingly complex N-glycan structures and functions in various organisms. The overall purpose of this research project has been to characterize the diversity within the α-mannosidase gene family of filamentous fungi. The α-1,2-mannosidases are involved in mannose removal in the intermediate stages of the N-glycosylation pathways, and diversification of this gene family may have provided the first significant divergence in these pathways among major lineages. Four novel α-mannosidases were identified and characterized from the filamentous fungus Aspergillus nidulans. These genes were designated Class II α-mannosidase, Class I α-1,2-mannosidase IA, Class I α-1,2-mannosidase IB and Class I α-1,2-mannosidase IC, based on their similarity to other Class I and Class II α-mannosidase sequences. The Class II α-mannosidase was highly similar to the rat ER/cytosolic and yeast vacuolar Class II α-mannosidases, and these three proteins formed a phylogenetically distinct subgroup, Class IIC. The Class I enzymes were highly related to each other, and to other fungal Class I α-1,2-mannosidases. Phylogenetic analysis indicates these genes duplicated and diverged subsequent to the divergence of fungi from insects and mammals. In addition to this research on A. nidulans, a single Class I α-1,2-mannosidase was identified and characterized from the Dutch Elm pathogen, Ophiostoma novo-ulmi, which was highly related to the A. nidulans Class I α-1,2-mannosidase IA and IC enzymes, and less so to the A. nidulans Class I α-1,2-mannosidase IB. Analysis of the function and/or biochemical properties of these enzymes was examined using several methods. Disruption and overexpression of the A. nidulans Class IIC α-mannosidase did not have any noticeable effect on the growth or morphology of the organism, indicating that this gene was not essential for growth. Biochemical characterization of the A. nidulans Class I α-1,2-mannosidase IC was initiated by recombinant secretion of the enzyme into culture media. Successful expression of the enzyme showed that the α-1,2-mannosidase IC did not exert any cytotoxic effects when overexpressed, suggesting that high levels of expression and purification should be feasible. Finally, disruption of the Class I α-1,2-mannosidase from O. novo-ulmi slightly altered the morphology of the organism, but was not lethal. The possible presence of multiple Class I α-1,2-mannosidases in this organism could explain the non-lethality of this mutation. Elucidation of the N-glycosylation pathways of A. nidulans may be useful in host strain improvement for heterologous protein expression systems. Modulation of the N-glycosylation pathways to produce specific N-glycan structures would increase the utility of the host for the production of human therapeutic glycoproteins which require these N-glycans for efficacy. Additionally, investigation of the genetic components of the N-glycosylation pathways of the Dutch Elm pathogen may provide global antifungal targets with broad applicability in other fungi. / Graduate

The principal aim of the study was to further the knowledge of the interaction between Alternaria linicola and the host plant linseed (Linum usitatissimum). A novel detached cotyledon in vitro bioassay was developed to allow the quantification of disease resistance of Linum material to A. linicola. Differences were apparent between the disease response scores of four linseed varieties when tested with seven isolates of the pathogen which differed in aggressiveness. However, there was no significant difference between the disease response scores of the varieties and no change in the ranking of varieties over three experiments. This indicated that the varieties behaved in a predictable manner to each isolate during each test. Accordingly, in a subsequent study, 102 Linum accessions were challenged with an aggressive and a non-aggressive isolate. About 75 % of the accessions gave a moderate response, although there was a continuous distribution of resistance from high susceptibility to resistant. Accessions at both extremes of the disease response consisted of breeding material, currently grown varieties and near relatives of the host species. For example, one of the more resistant accessions tested was Linum angustifolium. A sub-set of nine Linum accessions was chosen (a range of susceptible, moderately-resistant and resistant material) and the resistance response of the material to an aggressive and a non-aggressive A. linicola isolate was investigated using a whole seedling inoculation technique. A comparison of the response of the material during the seedling test with that of the detached in vitro assay indicated that the latter test systematically, but marginally, overestimated the disease response. The in vitro bioassay scores and the seedling test scores were positively correlated following inoculation with the more aggressive of the two isolates. It was suggested that the resistance response of material could be accurately predicted by the in vitro bioassay but that a certain level of isolate aggressiveness was necessary to differentiate between responses of the accessions. Since large isolate-line interactions with respect to resistance scores were not observed, the results implied that resistance was polygenically determined. These results indicate that the bioassay for disease resistance produces an accurate measure of resistance and provides plant breeders with a useful tool which can be utilised during breeding programs.

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1. The numbers and distribution of mating-type factors in local populations of Polystictus versicolor have been studied, and the data available on the number and distribution of mating-type alleles in other fungi have been reviewed. Possible explanations for the low numbers recorded in Polyfitictus versioolor are -given, and the potential out-breeding capacity of the local populations is discussed. 2. Growth rates of dicaryotic and monocaryotic strains of P. versicolor at 25°C and 35°C. have been studied. 3. Studies have been made on the genetical and physiological nature of the mycelium in P. versicolor and it is thought that the mycelium exists in nature as a physiologically homogenous and genetically heterogenous unit. The mechanisms by which such a mycelium could be established have been studied. They are (a) hyphal fusions and (b) dj. x mon. pairings. 4. The effects of different mating-type factors in compatible pairings on (a) hyphal fusion; (b) nuclear migrations; (c) clamp connection formations have been investigated, and a suggestion made concerning a model for gene action in connection with the formation of clamps.