Erwinia soft rot is a widespread disease destructive to numerous important crop plants. Damage to plants is primarily due to celldegrading enzymes (CDEs) secreted by the bacteria. I am interested in potato (Solanum tuberosum) soft rot because it is of agricultural importance and it represents an ideal model system for understanding molecular events in plant-pathogen interactions. Much has been learned in vitro about the molecular genetics of CDEs in the past decade; however, little is known about their expression in plantae To study expression of genes for these enzymes during pathogenesis and plant responses to erwinias or their enzymes, I developed a membrane-separated system for simultaneous studies of potato and bacterial gene expression. This system facilitates the isolation of plant tissue-free bacterial cells and bacteria-free plant tissue for subsequent analysis of gene expression by RNA blot hybridization. Using this system, I demonstrated that in compatible interactions, rnRNAs for three Erwinia carotovora subsp. carotovora (Ecc) CDE genes were induced to high levels and were induced sequentially: exo-pectate lyase (PL), endo-PL, and then endopolygalacturonase (PG) with maximal mRNA accumulations at 6, 9, and 12 hr, respectively. Induction of these mRNAs was well correlated with tissue maceration. In the incompatible interaction, however, induction of all three Ecc genes was reduced several-fold compared to the compatible interaction. The kinetics of mRNA accumulation during pathogenesis were distinct from those of in vitro accumulation induced by polygalacturonic acid. My results confirm that in planta expression of these genes was induced by exo-PL reaction products as suggested by other researchers. In studies of plant genes correlated with plant responses to pathogens and environmental stresses [plant defenseresponse (PDR) genes], I also showed Ecc triggered active responses distinct from wound responses. I used gene probes for phenylalanine ammonia- lyase (PAL) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), key genes in the biosynthesis of phenylpropanoid- and terpenoidderived compounds believed to be important in plant defenses. Ecc inoculation caused much more rapid and greater increases in PAL mRNA and enzyme activity levels in potato tuber than wounding alone. Escherichia coli, a non-plant pathogen, carrying a plasmid which encodes Ecc endo-PL, also induced PAL mRNA accumulation. Ecc induced a specific HMGR isogene (HMGR1) not activated by wounding. My results support the existence of an HMGR mul-ci-gene family. Wounding resulted in a rapid and transient accumulation of HMGR2 mRNA followed by a slower accumulation of HMGR3 mRNA. These isogenes are distinct from the Ecc-induced HMGRI gene. / Ph. D.

Environmental use of genetically-engineered microorganisms (GEMs) has raised concerns over potential ecological impact. Development of microcosm systems useful in preliminary testing for risk assessment will provide useful information for predicting potential structural, functional, and genetic effects of GEM release. This study was executed to develop techniques that may be useful in risk assessment and microbial ecology, to ascertain which parameters are useful in determining risk and to predict risk from releasing an engineered strain of Erwinia carotovora. A terrestrial microcosm system for use in GEM risk assessment studies was developed for use in assessing alterations of microbial structure and function that may be caused by introducing the engineered strain of E. carotovora. This strain is being developed for use as a biological control agent for plant soft rot. Parameters that were monitored included survival and intraspecific competition of E. carotovora, structural effects upon both total bacterial populations and numbers of selected bacterial genera, effects upon activities of dehydrogenase and alkaline phosphatase, effects upon soil nutrients, and potential for gene transfer into or out of the engineered strain. No significant difference was found in survival of the engineered strain as compared to its wildtype parent. Both strains survived for over two months in microcosms. The effects of both strains upon populations of total bacteria and selected bacterial genera were determined; while some effects upon community structure were observed, they were not significant. The engineered strain was not found to be a superior competitor compared to its parent; three different doses of engineered and wildtype strains were used. ln addition, neither strain affected activities of dehydrogenase or alkaline phosphatase in soil. Likewise, no effects were observed upon the nutrients monitored. However, transfer of the kanamycin resistance gene that had been inserted into the engineered E. carotovora strain may have occurred. Five species of indigenous bacteria displayed kanamycin resistance 15 days after being exposed to the engineered Erwinia. DNA from these strains was isolated, purified, and hybridization experiments executed to determine if any homology existed between these DNAs and the kanamycin resistance gene that had been inserted into E. carotovora. Using biotin-Iabeled probes and Iow-stringency washing conditions, homology was observed. However, before gene transfer can be proven, additional studies, including amplification and sequencing, may be required. Although a simple microcosm design was employed, it yielded sufficient information to conclude that release of the engineered Erwinia carotovora will not affect any of the microbial measures of integrity that were studied in a manner different from that of the wildtype. Effects upon plant material and other higher taxa will be the focus of future studies. / Ph. D.

A mandioquinha-salsa (Arracacia xanthorriza Bancroft.) é uma raiz tuberosa que apresenta um curto período de conservação pós-colheita, de 3 a 5 dias, devido a uma fitopatologia conhecida como apodrecimento-mole ou mela, causada por bactérias do gênero Erwinia. Essas bactérias liberam enzimas que degradam a pectina da parede celular, fazendo com que o tecido perca a sua rigidez característica. Atualmente, vários métodos de conservação têm sido estudados na tentativa de prolongar a conservação pós-colheita, porém, a combinação de processos parece ser a melhor alternativa. Esse trabalho teve como objetivo estudar a interação entre processos de conservação (refrigeração, embalagem a vácuo e irradiação) para estender o período pós-colheita das raízes. Foram estudadas a combinação de duas temperaturas (25°C e 4°C), duas embalagens (caixas e vácuo) e três doses de irradiação gama obtendo um total de 16 grupos. Estes foram analisados diariamente, por um período de 30 dias, utilizando parâmetros de textura (energia de penetração), microbiologia e atividade de enzimas pectinolíticas (pectato liase, poligalacturonase e pectinesterase). A exposição às doses de 2 e 3kGy, com as amostras conservadas a 4°C a vácuo, prolongaram o período de conservação de 5 para 28 dias, ocorrendo uma diminuição da população microbiana, porém havendo uma diminuição da rigidez das raízes (p<0,05). Os tratamentos afetaram o perfil de atividade das enzimas pectinolíticas, porém a grande dispersão dos resultados e o pequeno número de raízes analisadas por dia, além da complexidade dos fatores que afetam a atividade das enzimas e as múltiplas origens possíveis - endógenas, bacterianas ou fungicidas - limitam a discussão mais aprofundada dos resultados. / Peruvian carrot (Arracacia xanthorriza Bancroft.) is a tuber root that presents a short post-harvest period of conservation, 3 to 5 days, due to a phytopathology known as soft rot or \"mela\", caused by bacteria of the genus Erwinia. This bacteria release enzymes that decay the cellular wall, causing the lost of the characteristic rigidity. At present, many conservation methods have been studied in the attempt of prolonging the post harvest conservation, but the combination of processes seems to be the best alternative. The aim of this work was to study the interaction between the conservation processes (refrigeration, vacuum packaging and irradiation) to extend the post-harvest period of the roots. It was studied the combination of two temperatures (25°C e 4°C), with two packages (boxes and vacuum) and three gamma irradiation doses (1, 2 e 3kGy), obtaining a total of 16 sample groups. The samples were daily analized, for a 30 day period, using texture parameters (penetration energy), microbiology and pectinolitic enzymes activities (pectate lyase, polygalactunoronase and pectin methyl esterase). The samples irradiated in doses of 2 and 3kGy, vacuum packed and conserved at 4°C extend the post-harvest period of 5 to 28 days, with a decrease of the microbiologic population, but with decreased in the rigidity of the roots (p<0.05). The treatments affected the pectinolitic enzymes profile, however the amplitude of the results and the low number of analysed samples per day, besides the complexity of factors affecting the enzyme activity and the multiple possible sources(endogenous, bacterial or fungous), limits the carefully discussion of the results.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2008. / Härtill 4 uppsatser.

Thèse (Ph. D.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 9 février 2006). Bibliogr.

A mandioquinha-salsa (Arracacia xanthorriza Bancroft.) é uma raiz tuberosa que apresenta um curto período de conservação pós-colheita, de 3 a 5 dias, devido a uma fitopatologia conhecida como apodrecimento-mole ou mela, causada por bactérias do gênero Erwinia. Essas bactérias liberam enzimas que degradam a pectina da parede celular, fazendo com que o tecido perca a sua rigidez característica. Atualmente, vários métodos de conservação têm sido estudados na tentativa de prolongar a conservação pós-colheita, porém, a combinação de processos parece ser a melhor alternativa. Esse trabalho teve como objetivo estudar a interação entre processos de conservação (refrigeração, embalagem a vácuo e irradiação) para estender o período pós-colheita das raízes. Foram estudadas a combinação de duas temperaturas (25°C e 4°C), duas embalagens (caixas e vácuo) e três doses de irradiação gama obtendo um total de 16 grupos. Estes foram analisados diariamente, por um período de 30 dias, utilizando parâmetros de textura (energia de penetração), microbiologia e atividade de enzimas pectinolíticas (pectato liase, poligalacturonase e pectinesterase). A exposição às doses de 2 e 3kGy, com as amostras conservadas a 4°C a vácuo, prolongaram o período de conservação de 5 para 28 dias, ocorrendo uma diminuição da população microbiana, porém havendo uma diminuição da rigidez das raízes (p<0,05). Os tratamentos afetaram o perfil de atividade das enzimas pectinolíticas, porém a grande dispersão dos resultados e o pequeno número de raízes analisadas por dia, além da complexidade dos fatores que afetam a atividade das enzimas e as múltiplas origens possíveis - endógenas, bacterianas ou fungicidas - limitam a discussão mais aprofundada dos resultados. / Peruvian carrot (Arracacia xanthorriza Bancroft.) is a tuber root that presents a short post-harvest period of conservation, 3 to 5 days, due to a phytopathology known as soft rot or \"mela\", caused by bacteria of the genus Erwinia. This bacteria release enzymes that decay the cellular wall, causing the lost of the characteristic rigidity. At present, many conservation methods have been studied in the attempt of prolonging the post harvest conservation, but the combination of processes seems to be the best alternative. The aim of this work was to study the interaction between the conservation processes (refrigeration, vacuum packaging and irradiation) to extend the post-harvest period of the roots. It was studied the combination of two temperatures (25°C e 4°C), with two packages (boxes and vacuum) and three gamma irradiation doses (1, 2 e 3kGy), obtaining a total of 16 sample groups. The samples were daily analized, for a 30 day period, using texture parameters (penetration energy), microbiology and pectinolitic enzymes activities (pectate lyase, polygalactunoronase and pectin methyl esterase). The samples irradiated in doses of 2 and 3kGy, vacuum packed and conserved at 4°C extend the post-harvest period of 5 to 28 days, with a decrease of the microbiologic population, but with decreased in the rigidity of the roots (p<0.05). The treatments affected the pectinolitic enzymes profile, however the amplitude of the results and the low number of analysed samples per day, besides the complexity of factors affecting the enzyme activity and the multiple possible sources(endogenous, bacterial or fungous), limits the carefully discussion of the results.

Le but de cette thèse était d’évaluer le potentiel antimicrobien de 21 sels organiques et inorganiques pour contrôler la pourriture molle de la pomme de terre, causée par les bactéries Erwinia carotovora subsp. carotovora et Erwinia carotovora subsp. atroseptica. Les résultats obtenus ont montré que 11 sels ont complètement inhibé la croissance bactérienne in vitro, parmi lesquels le carbonate de sodium, le métabisulfite de sodium, le phosphate de sodium tribasique, le lactate d’aluminium, le chlorure d’aluminium, le bicarbonate de sodium, l’acétate d’ammonium, le di-hydroxy acétate d’aluminium, le sorbate de potassium et le benzoate de sodium se sont avérés bactéricides. Appliqués sur les tubercules de pomme de terre, seuls le chlorure d’aluminium, le métabisulfite de sodium et dans une moindre mesure le benzoate de sodium ont permis de réduire la sévérité de la maladie, et ce de façon curative et préventive. Il semble à la lumière des résultats obtenus, que l’effet inhibiteur des sels observé in vitro résulte de la capacité d’ionisation de l’eau (faible pKa ou pKb) de leurs constituants, ainsi que de leur lipophilicité (dans le cas du benzoate de sodium et du sorbate de potassium). Cette capacité d’ioniser l’eau des ions serait cependant altérée par le pouvoir tampon du tubercule. Pour leur part, les sels efficaces contre la maladie disposeraient de caractéristiques spécifiques leur permettant de demeurer actifs dans le tissu. Des études ultrastructurales ont montré que le chlorure d’aluminium provoque le bris de l’enveloppe bactérienne, une disparition des vésicules extracellulaires et une agrégation du cytoplasme, phénomènes non observés chez les bactéries traitées avec le métabisulfite de sodium. Enfin, l’évaluation de l’effet de certains sels sur la qualité des tubercules a montré d’une part que la perte de poids a été généralement plus importante chez les tubercules traités au chlorure d’aluminium ou au métabisulfite de sodium, et d’autre part que l’application des sels organiques, particulièrement de benzoate de sodium, a favorisé une augmentation du contenu en sucres solubles chez les tubercules. Cette étude démontre que le chlorure d’aluminium et le métabisulfite de sodium pourraient être mis à profit pour la conservation de la pomme de terre. / The objective of this thesis was to evaluate the antimicrobial potential of 21 organic and inorganic salts to control Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), two bacteria responsible for soft rot development in stored potato tubers. The results have shown that eleven salts (at 0.2 M) have completely inhibited the bacterial growth in vitro, among which sodium carbonate, sodium metabisulfite, trisodium phosphate, aluminum lactate, aluminum chloride, sodium bicarbonate, ammonium acetate, aluminum di-hydroxy-acetate, potassium sorbate and sodium benzoate were bactericidal. On potato tubers, only aluminum chloride, sodium metabisulfite and to a lesser extent sodium benzoate have markedly controlled the disease severity, during both curative and preventive applications. Based on the obtained results, it appears that the inhibitory effect of salts in vitro relates to the water ionization capacity (low pKa or low pKb) of their constituent ions, as well as their lipophilicity. However, the effect was dampened in vivo, presumably due to the buffering capacity of tuber tissue and Donnan effect which could lead to generation of less effective ionic species. It is likely that those salts effective in vivo exhibit other properties contributing to their effectiveness. Ultrastructural studies on Eca showed that aluminum chloride caused rupture of bacterial envelope, and cytoplasmic aggregation, which were not observed in the bacteria treated with sodium metabisulfite. These observations suggest that a part of the toxic effect of aluminum chloride originates from alterations of the bacterial envelope, whereas rapid bacterial death caused by sodium metabisulfite occurs intracellularly through interaction with biomolecules, facilitated by the diffusion of SO2. Finally, evaluation of the effect of salts on tuber quality revealed that tuber weight loss was generally higher with aluminum chloride and sodium metabisulfite treatments, while the organic salts, particularly sodium benzoate, increased sugar content of the tubers. It was concluded that aluminum chloride and sodium metabisulfite can be profitably used to fight potato storage soft rot.

The ability of a genetically-engineered <i>Erwirzia carotovora</i> subsp. <i>carotovora</I> (Ecc) strain to transfer recombinant chromosomal DNA or plasmids to wildtype Ecc or <i>Pseudomonas fluorescens</i> was tested on filters, within soil microcosms, and <i>in planta</i>. Ecc was engineered by chromosomal insertion of a disarmed <i>endo</i>-pectate lyase gene marked with a 1.4kb DNA fragment conferring kanamycin resistance. Plasmids RPI and pBR322 were introduced separately into engineered Ecc clones. These strains served as donors in genetic transfer experiments. No transfer of the inserted kan marker or of pBR322 was observed under any experimental condition. In filter matings, RPI was transferred to wildtype Ecc at a frequency of 3.6 X 10⁻² transconjugants per donor (TPD) and to P. <i>fluorescens</i> at a frequency of 2.4 X 10⁻⁵ TPD. In matings conducted in potato tubers inoculated using sewing needles, the respective frequencies were 4.0 X 10⁻³ and 2.0 X 10⁻³, while matings on potato slices yielded frequencies of 4.7 X 10⁻² and 2.3 X 10⁻². In soil microcosms, the maximum transfer frequencies observed were 2.3 X 10³ and 8.4 X 10⁻⁵ TPD. / Master of Science