Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 126-135).

Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 110-118).

Factors affecting a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Erwinia carotovora subsp. atroseptica were investigated. Optimum reaction conditions for detecting known cell numbers of Eca were found to be 2.μg/ml of coating γ-globulin and 1:4-00 enzyme-γ-globulin conjugate dilution. These conditions were determined using antiserum produced against glutaraldehyde-fixed, whole bacterial cells of strain E82 of Eca (serogroup I), and polystyrene microtitration plates (Dynatech substrate plates). In spite of these optimized conditions, variability was observed between sets of data obtained under identical experimental conditions. In order to minimize or eliminate this variability, different parameters were investigated. The washing procedure was standardized by the use of a controlled pressure-washing system employing distilled water, and two 15-sec washes at 34.4-8 kPa (5 psi), with 180° rotation of the plate between each wash. Tween-20 was eliminated from the washing solution, since it interferred with the sensitivity of the assay. This effect could not be related to the age of the Tween-20 employed. Well to well variability was observed with the polystyrene microtitration plates employed but it was not exclusive to the outside rows. The pattern of distribution of the "odd" wells within a plate changed, and the number of "odd" wells decreased with time. The maximum variation from the mean also decreased with time. Addition to different wells of an extra 5% of coating y-globulin, sample, and enzyme-y-globulin conjugate individually or in different combinations, failed to reproduce the variability observed thereby eliminating pipetting errors as a source of variability. The A[sub=+.05] values were influenced by the buffer solutions employed for sample and conjugate dilution. Any given buffer had a greater effect when used for conjugate dilution. The complete buffer of phosphate buffered saline (PBS)+ 0.05% Tween-20 +2.0% polyvinylpyrrolidone+0.2% egg albumin commonly used in virus work, was found to be suitable for the Eca system although its efficiency in the presence of plant material containing bacteria remains to be evaluated. This ELISA for Eca employing optimized coating and conjugate, a standardized washing procedure and a complete buffer for samples and conjugate dilution, routinely detected 10⁵ to 10⁶ cells/ml of only serogroup I of Eca when pure cultures of both homologous and heterologous strains were tested. At concentrations >10⁷ cells/ml, strains from serogroups XVIII, XX, and XXII of subsp. atroseptica and a few strains from serogroups II, III, IV, and V of subsp. carotovora also reacted. Even at high bacterial concentration (10° cells/ml) no cross reactions were observed with Pseudomonas marginalis and Corynebacterium sepedonicum. Heat treatment of cell suspensions of serogroup I at 60 C for 3-6 min enhanced A[sub=+.05] values but the level of sensitivity was not reduced below 105 cells/ml. Cross reactions with strains of subsp. caro- tovora serogroups III and V, observed at 10 cells/ml, were reduced but not eliminated by this heat treatment. Both heat-labile and heat-stable water-soluble antigens were detected by this ELISA for Eca. The media upon which cells were grown also affected the A[sub=+.05] values but this effect was not proportional to the amount of growth observed. Based on these results it was concluded that until well to well variability is eliminated, and sensitivity increased, there will be little incentive to use the double sandwich ELISA technique with plant sap where a reduction in sensitivity is likely. At this point ELISA seems to have little potential in routine surveys for detecting latent blackleg infections in certified seed potatoes. / Land and Food Systems, Faculty of / Graduate

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Erwinia strains from several geographic regions and several hosts were evaluated for virulence, sensitivity to siderophores produced by Pseudomonas fluorescens (Pf) and bacteriocins produced by Erwinia, and for the presence of plasmids. Selection of virulent strains of Erwinia for use in plant breeding programs for resistance to disease might be based upon quantitative characters which are correlated with disease severity rather than the biochemical reactions used to distinguish Erwinia carotovora subspecies. Quantitative assays for motility, polygalacturonate degradation, potato tuber infection, and tuber decay revealed that motility was correlated with infection (r = 0.83, p = 0.01) and polygalacturonate degradation with decay (r = 0.84, p = 0.01) of potato tubers. Siderophores produced by Pf and bacteriocins produced by Erwinia yielded variable results in bioassays against the Erwinia strains studied. Six of the 12 strains of Erwinia tested were resistant to Pf siderophores. The growth inhibition of sensitive strains was bacteriostatic and reversible by addition of iron (Fe II or Fe III) to the culture medium. Additionally, only one strain of Erwinia was sensitive to the bacteriocins produced by the other 12 strains. The resistance of Erwinia strains to Pf siderophores and Erwinia bacteriocins severely limits the potential for widespread application of these agents in biological control of Erwinia. Bacteriocin-like structures were detected in culture extracts of all 12 Erwinia strains studied. The presence of bacteriocins is indirect evidence that these strains harbor plasmids. Bacteriocin-coding plasmids may be the source of genetic and phenotypic variability demonstrated by the erwinias. The status and value of subspecific classification of Erwinia carotovora may therefore require re-evaluation.

Call number: LD2668 .T4 1984 A4 / Master of Science

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Entomology." Includes bibliographical references (p. 132-139).

No description available.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1975.

Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40 percent by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9 percent confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P.