Thesis (Ph. D.)--University of Wisconsin--Madison, 1976. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves 97-108.

The extent of tuber contamination with Erwinia carotovora in B.C. grown seed potatoes was determined by incubating tubers in a moist, anaerobic atmosphere. Only 2.1% and 11.3% of the tubers indexed in 1972 and 1975, respectively, were found to be latently-infected with 15. carotovora. Recoveries within individual lots ranged from 0 to a high of 27.7%. Both var. carotovora and var. atroseptica were recovered from tubers with equal frequency. No correlation was found between the field incidence of blackleg and the degree of latent tuber infection. Representative isolates of both varietal forms were able to infect faba beans, cucumbers and peppers while none was able to infect cereals, forage legumes or common weeds like black nightshade, horsetail, and lamb's quarters. Approximately 50% of the var. carotovora isolates recovered in the surveys incited blackleg symptoms in stem-prick inoculated Erwinia-tested potato stem cuttings grown at 18° C. Pathogenic carotovora isolates were also found to have a much wider host range than the nonpathogenic carotovora isolates. The additional hosts expressing symptoms were onions, peas, petunias, tobacco, tomato, lady's thumb and Chenopodium quinoa L. The majority of the pathogenic isolates (73%) reacted with an antiserum prepared against a pathogenic var. carotovora isolate. The atroseptica varietal form was found not to survive in artificially infested fallow field soils beyond 8 weeks under winter conditions and 4 weeks under summer conditions. By contrast var. carotovora was recovered from similarly infested soil at the conclusion of the overwintering experiments (139 and 225 days). The carotovora form also survived 29 days longer in soils in the oversummering experiment. The atroseptica form could not be detected around inoculated seed pieces after 4 weeks. However, var. carotovora was still recovered from soil in 2 out of 5 replicates at the end of the experiment (70 days). Both pathogenic and nonpathogenic isolates of the carotovora form were easily recovered from the rhizosphere of horsetail, lady's thumb, lamb's quarters, nightshade and redroot pigweed during the fall but rarely in similar surveys during the spring. The atroseptica form was recovered only once, from the rhizosphere of a horsetail plant during the September 1974 survey. The atroseptica form could still be recovered from artificially infested rhizospheres of lamb's quarters at the end of the experiment (140 days) but was not detected in the rhizospheres of lady's thumb after 42 days or in the rhizospheres of horsetail after 112 days. Parallel experiments with var. carotovora indicated that while this varietal form could survive in soils, weed rhizospheres help to maintain a high inoculum potential. Survival on tubers was found to be the only way in which var. atroseptica could survive winter in numbers which were detectable the following spring. Recovery f rom tubers buried 10 cm deep was greater than from those on the surface after 225 days. Surface-borne tubers inoculated with the atroseptica form tended to decay more rapidly which accounted for the lower recovery rate. The survival of var. carotovora was less dependent upon burial of tubers but the recovery rates were generally higher if tubers were buried. / Land and Food Systems, Faculty of / Graduate

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The sensitivity of Erwinia carotovora subsp. atroseptica to bacteriocins produced by strains in the common potato serogroups of E. carotovora was investigated. Bacteriocins produced by representative strains of the common serogroups had activity spectra containing strains from one to six sensitive serogroups. Similarly, indicator strains representing different serogroups showed variable sensitivity. One indicator strain was sensitive to bacteriocins produced by only one producing strain while others were sensitive to bacteriocins produced by strains in several different serogroups. Bacteriocin production in the serogroups tested was detected only from strains that were biochemically E.c. subsp. carotovora (Ecc). Strains in all four E.c. subsp. atroseptica (Eca) serogroups were bacteriocin sensitive and non-producers. Some Ecc strains were bacteriocin producers and sensitive to bacteriocins produced by strains in other serogroups. Production and sensitivity were not correlated with the frequency of distribution of the more common serogroups isolated in nature. Representative strains in the two most common serogroups (I and III) were sensitive to bacteriocins produced by representative strains in three and six serogroups respectively. Strains in some of the less common serogroups (IX, XI and XVI) were bacteriocin producers but were not sensitive to the bacteriocins produced by the representative strains tested. Thus, a role for bacteriocins in the survival of these strains in nature cannot be ruled out. Of the 44 serogroup XI strains tested by the agar overlay technique, 31 were "typical producers", 10 were "differential producers" and only three were "non-producers". However, bacteriocin production in the latter group could be detected after induction with Mitomycin C but not with UV light. In the five serogroups in which several strains were tested, bacteriocin production and sensitivity were serogroup rather than subspecies characteristics. In dual culture studies the starting ratio of "typical producer" to sensitive strains of 1:1000 prevented detectable growth of the sensitive strains. By comparison a starting ratio of 100:1 with a "non-producer" strain did not prevent growth of the sensitive strains. Similar results were obtained when potato tuber discs were inoculated with varying starting ratios and the population monitored after 48 h. Thus, bacteriocin producing strains have a selective advantage when grown together in vitro with the bacteriocin sensitive, non-producing strains. Bacteriocin titres were enhanced by Mitomycin C induction and partial purification. Following ammonium sulfate precipitation and ultracentrifugation (150 000 x g for 90 min), bacteriocin activity in the resuspended pellet was associated with particles which by transmission electron microscopy resembled contractile, bacteriophage tail-like particles. These particles (due to their molecular size) were associated with small (≈ 4 mm) clear zones of inhibition in the spot assay tests. "Bacteriocin-like" activity in the supernatant was resolved by gel filtration into three fractions with estimated molecular weights of 17 700, 29 500 and 224 000 D. The first two fractions showed large (up to 20 mm) diffuse zones of inhibition. The third fraction showed small (≈ 4 mm) clear zones of inhibition. All four fractions had similar activity spectra against representative indicator strains and were produced by all of the serogroup XI producer strains tested. Relative production differed depending on the strain. The threshold of sensitivity displayed by the indicator strains varied with the fractions. The resuspended pellets had the highest titres which suggested that those macromolecular bacteriocins were responsible for the antagonism in in vitro and possibly in nature. / Land and Food Systems, Faculty of / Graduate

Graduation date: 1993

Thesis (M.S.)--University of Wisconsin--Madison, 1969. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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