Thesis (Ph. D.)--Swedish University of Agricultural Sciences, 2001. / Includes bibliographical references.

The habitat diversity of the fungus Nectria haematococca MPVI has been shown to be due in part to conditionally dispensable (CD) chromosomes that carry habitat-defining genes. From a biological perspective, the CD chromosomes are analogous to plasmids that possess genes that determine the habitats of plant-associated bacteria. This study establishes that the N. haematococca CD chromosome that contains the genes for Pea Pathogenicity (PEP cluster) also carries genes for the utilization of homoserine, an amino acid found in pea root exudates. Competition studies presented here demonstrate that an isolate that lacks the PEP cluster, but carries a portion of the CD chromosome containing the homoserine utilization (HUT) genes, is more competitive in the pea rhizosphere than an isolate without the CD chromosome. Further competition studies show that both the PDA1 and PDA6 CD chromosomes confer a competitive advantage in the rhizosphere of soybean, whereas only the PDA6 CD chromosome confers a competitive advantage in the rhizospheres of tomato and alfalfa, and only the PDA1 CD chromosome confers a competitive advantage in the rhizosphere of pea. These studies suggest the presence of genes on the PDA6 and PDA1 CD chromosomes that enhance the ability of N. haematococca to expand its habitat and support the idea that fungal CD chromosomes are analogous to host-specifying plasmids in plant-associated bacteria. Transformation, insertional mutagenesis, and bioinformatics were used to identify a cluster of five genes on the PDA1 CD chromosome that was responsible for the HUT phenotype in N. haematococca. One of the genes was found only in N. haematococca, another was a fungal transcription factor, and the other three had homologs involved in the synthesis of the amino acids methionine, threonine, and isoleucine, in which homoserine is an intermediate. Competition experiments that compared isolates with or without the HUT cluster showed that the HUT cluster is responsible for increased competitive ability of HUT+ N. haematococca isolates in the rhizosphere of pea. This study establishes that homoserine utilization can be a rhizosphere competency trait for N. haematococca and, to our knowledge, is the first example of a rhizosphere competency trait identified in a fungus.

Platelets are small fragments, but they are of crucial importance for the coagulation. The risk of spontaneous bleeding increases when the level of platelets falls below a thrombocyte particle concentration threshold value of 50 x 109/L. In those cases a platelet transfusion might be compulsory. Ongoing research tries to improve the quality of the platelets and to increase the safety of the method used. However, we still need to better understand which factors that affect how patients react upon platelet transfusion. In this study, 100 transfusions performed at Uppsala University Hospital during 2009 were examined. The platelets used had been produced with apheresis followed by pathogen inactivation by Intercept Blood SystemTM. Platelets were counted before and after transfusions and the increase was calculated in purpose to examine how well the patients responded to the platelet transfusions. These values were plotted against platelet storage time in order to examine the possible impact on the result of treatment.

Emerging infectious diseases, pollution and climate change are associated with amphibian extinction events worldwide; however, direct causation is often obscured by the interactions of these stressors. Elucidating the possible synergies between metal contamination and disease is, therefore, critical in advancing our knowledge of the co-evolutionary mechanisms in host-pathogen systems and helping with the ability to better forecast the spread of diseases in metal-stressed environments. Additionally, increasing ecotoxicological research has improved our understanding of the complex influence trace metals may have on the physico-chemical nature of aquatic systems; however, the discrepancy in concentration-response within the toxicological literature makes it difficult to accurately define the range of toxicity, often due to the variability in media used in experimentation. The first chapter of this thesis reports an evaluation of copper, nickel and copper/nickel concentrations on several Northern Leopard Frog (Lithobates [Rana] pipiens) larvae life history traits within field collected smelting effluent water. Overall, results indicated that copper had a stronger negative impact on survival than nickel. However, tadpoles exposed to copper displayed increased growth and developmental patterns while those exposed to nickel demonstrated opposing life history traits. These results indicate that tadpoles are displaying different fitness strategies, in terms of survival and life history, in the presence of increased copper and/or nickel stress.

Arbuscular mycorrhizal fungi (AMF) are widespread soil dwelling microorganisms that associate with plant hosts. AMF receive carbon from the host as a result of the mutualism, while the plant’s ability to acquire nutrients is enhanced by AMF. Additionally, AMF benefit their host in the form of pathogen protection. While it is known that increased AMF species richness positively correlates with aboveground plant productivity, the relationship between AMF diversity and pathogen protection is not well understood. In a growth chamber study, the plant host Leucanthemum vulgare, a non-native plant species in North America, was introduced to all combinations of three AMF species either in the presence or absence of the plant root pathogen Rhizoctonia solani. In the presence of the pathogen, the plant host increased its dependence on the AMF symbiosis. However, the richest AMF species assemblage did not provide the greatest pathogen protection. Understanding how diverse groups of AMF protect plants from pathogen attack provides insight into how plant communities are formed and structured. / NSERC

Uninfected larvae of Ixodes dammini, lxodes cookei, and Amblyomma americanum were fed on hamsters that had been injected intraperitoneally with a 0.5.ml sample of Borrelia burgdorferl (2.5 X 107 spirochetes per ml) 21 days earlier. A total of 108 of these larvae comprised of 36 1. dammini, 36 i. cookei, and 36 A. americanum were aseptically dissected and examined by darkfield and immunofluorescent microscopy for the presence of B. burgdorferl within 48 hours of feeding on the B. burgdorferi infected hamsters. The removal and examination of the midgut diverticula revealed that 32/36 (88.9%) of the l. dammini larvae contained B. burgdorferl. Only 5/36 (13.9%) of the l. cookei larvae and 7/36 of the A. americanum larvae harbored spirochetes in their midgut diverticula.A portion of the nymphs that molted from the above larvae were also dissected and examined by darkfleld and indirect immunofluorescent techniques. Borrelia burgdorferi were observed in the midgut diverticula of 94/107 (87.8%) of the l. dammini nymphs. None of the 30 (0%) l. cookei nymphs examined were found positive for spirochetes and only 1/60 (1.7%) of the A. americanum nymphs was found positive for B. burgdorrerl.A total of 83 lL dammini, 53 A. americanum, and 161. cookei nymphs reared from larvae that fed to repletion on hamsters infected with B. burgdorrerl were allowed to feed on uninfected hamsters to assess transmission of B. burgdorrerl. Transmission was demonstrated only by the l. dammlnl nymphs. The findings of this study suggest that it is extremely unlikely that l. cookei can serve as a vector for B. burgdorrerl, but do not rule out completely the possibility that A. americanum may be able to maintain B, burgdorrerl infections transstadially and, under certain conditions, transmit the organisms to vertebrate hosts. / Department of Physiology and Health Science

Uninfected nymphs of Ixodes scapularis and Amblyomma americanum were fed on hamsters that had been injected intraperitoneally with a 0.5 ml sample of Borrelia burgdorferi (2.5 X 10' spirochetes per ml) 30 days earlier. All nymphs fed to repletion and were then housed during the molting process. In order to assess their ability to transmit the spirochetes, the resulting l. scapularis and A. americanum adults were allowed to feed on uninfected rabbits.Dissection of the adult l. scapularis ticks revealed 9/12 (75%) to harbor motile spirochetes, identified as B. burgdorferi by darkfield microscopy, isolation in BSK II medium, and indirect immunofluorescent antibody staining with the monoclonal antibody H5332. Transmission was successful to one of two New Zealand White rabbits by these infected ticks.Dissection of the adult A. americanum ticks revealed 0/150 (0%) to harbor spirochetes. Transmission to each of three rabbits was unsuccessful. However, 5/90 (5.6%) cultures of midgut material from these same ticks, harbored non-motile spirochete-like bodies. The identity of these "spirochetes" is unknown. / Department of Biology

Polyadenylation of pre-mRNAs is critical for efficient nuclear export, stability, and translation of the mature mRNAs, and thus for gene expression. The bulk of pre-mRNAs are processed by canonical nuclear poly(A) polymerase (PAPS). Both vertebrate and higher-plant genomes encode more than one isoform of this enzyme, and these are coexpressed in different tissues. However, in neither case is it known whether the isoforms fulfill different functions or polyadenylate distinct subsets of pre-mRNAs. This thesis shows that the three canonical nuclear PAPS isoforms in Arabidopsis are functionally specialized owing to their evolutionarily divergent C-terminal domains. A moderate loss-of-function mutant in PAPS1 leads to increase in floral organ size, whereas leaf size is reduced. A strong loss-of-function mutation causes a male gametophytic defect, whereas a weak allele leads to reduced leaf growth. By contrast, plants lacking both PAPS2 and PAPS4 function are viable with wild-type leaf growth. Polyadenylation of SMALL AUXIN UP RNA (SAUR) mRNAs depends specifically on PAPS1 function. The resulting reduction in SAUR activity in paps1 mutants contributes to their reduced leaf growth, providing a causal link between polyadenylation of specific pre-mRNAs by a particular PAPS isoform and plant growth. Additionally, opposite effects of PAPS1 on leaf and flower growth reflect the different identities of these organs. The overgrowth of paps1 mutant petals is due to increased recruitment of founder cells into early organ primordia whereas the reduced leaf size is due to an ectopic pathogen response. This constitutive immune response leads to increased resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis and reflects activation of the salicylic acid-independent signalling pathway downstream of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)/PHYTOALEXIN DEFICIENT4 (PAD4). Immune responses are accompanied by intracellular redox changes. Consistent with this, the redox-status of the chloroplast is altered in paps1-1 mutants. The molecular effects of the paps1-1 mutation were analysed using an RNA sequencing approach that distinguishes between long- and short tailed mRNA. The results shown here suggest the existence of an additional layer of regulation in plants and possibly vertebrate gene expression, whereby the relative activities of canonical nuclear PAPS isoforms control de novo synthesized poly(A) tail length and hence expression of specific subsets of mRNAs. / Polyadenylierung von prä-mRNAs ist entscheidend für den Export aus dem Zellkern, die Stabilität und die Translation der reifen mRNAs und dadurch für die Genexpression. Der Großteil der mRNAs wird durch sogenannte canonische Poly(A) Polymerasen (cPAPS) prozessiert. Die Genome von sowohl Wirbeltieren als auch Pflanzen kodieren mehr als eine Isoform dieser Enzyme, welche gleichzeitig in verschiedenen Geweben exprimiert werden. Es ist jedoch kein Beispiel bekannt, das zeigt, ob die verschiedenen Isoformen unterschiedliche Funktionen einnehmen bzw. verschiedene Untergruppen von mRNAs polyadenylieren. Diese Arbeit zeigt, dass drei canonische PAPS Isoformen in Arabidopsis thaliana aufgrund ihrer evolutionär unterschiedlichen C-terminalen Domänen spezialisierte Funktionen haben. Eine schwache Verlust-Mutation im PAPS1 Gen bewirkt eine Vergrößerung der Blütenorgane, während die Blattgröße vermindert ist. Eine starke Verlust-Mutation bewirkt zusätzlich einen Defekt der männlichen Keimzellen. Im Gegenzug dazu sind Mutanten des PAPS2 oder PAPS4 Gens gesund und zeigen ein normales Wachstum. Polyadenylierung von SMALL AUXIN UP RNA (SAUR) mRNAs hängt spezifisch von der Funktion von PAPS1 ab. Die daraus entstehende Reduzierung der SAUR Aktivität in den paps1 Mutanten trägt zur Verringerung der Blattgröße bei und stellt eine kausale Verbindung zwischen Polyadenylierung spezifischer mRNAs durch bestimmte PAPS Isoformen und Pflanzenwachstum dar. Zusätzlich spiegeln die unterschiedlichen Effekte von PAPS1 auf Blüten und Blätter die Identitäten dieser Organe wieder. Das übermäßige Wachstum der mutanten Petalen beruht auf einer erhöhten Anzahl an Gründer-Zellen im frühen Primordium, wohingegen die verminderte Blattgröße auf eine ektopische Pathogen Antwort zurückzuführen ist. Diese konstitutive Immunantwort bewirkt eine erhöhte Resistenz der Mutanten gegenüber dem biotrophen Oomyceten Hyaloperonospora arabidopsidis und reflektiert die Aktivierung des Salizylsäure unabhängigen Signalweges von ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)/PHYTOALEXIN DEFICIENT4 (PAD4). Immunantworten sind von Veränderungen des intrazellulären Redoxpotenzials gekennzeichnet. Damit übereinstimmend zeigen die Chloroplasten der paps1-1 Mutanten ein verändertes Redoxpotenzial. Zur genaueren Aufklärung der molekularen Effekte der paps1 1 mutation wurde eine RNA-Sequenzierungsmethode verwendet, die zwischen mRNAs mit langem oder kurzem Poly(A) Schwanz unterscheidet. Die Aktivitäten der verschiedenen canonischen PAPS Isoformen kontrollieren die Länge des neu synthetisierten poly(A) Schwanzes und damit die Expression spezifischer Untergruppen von mRNAs. Dadurch lassen die hier gezeigten Ergebnisse eine weitere Ebene der Genregulierung in Pflanzen, und möglicherweise auch in anderen Eukaryoten, vermuten.

The bacterium Helicobacter pylori is a human pathogen that infects a large proportion of the world's population and is associated with serious diseases such as gastric ulcers and adenocarcinoma. The motility of this organism, by virtue of sheathed polar flagella is essential to colonisation and persistence in the human host. The sequencing of the H. pylori genome in 1996 identified homologues of the majority of the flagellar genes found in S. enterica serovai typhimurium. These included genes encoding the flagellum ATPase, FliI and FliH a presumptive inhibitor, the primary focus of this study. Sequencing did not originally identify an H. pylori homologue of the flagellar chaperone FliJ, and this is also considered in this study. Bioinformatic analysis and modeling suggests a structural and functional relationship between FliI and homologues such as F1-ATPase α- and β-subunit. In particular, residues 2-91 of FliI resemble the N-terminal domain of the F1-ATPase α- and β-subunits. Biochemical analyses reported in this thesis showed that a truncated FliI-(2- 91) protein was folded, although the N-terminal 18 residues were likely unstructured. Furthermore, deletion mutagenesis showed that this disordered segment of the protein mediates interaction with FliH and very likely forms an amphipathic α-helix upon forming of the FliI-FliH complex. The scanning mutagenesis of this interaction segment of FliI identified a cluster of conserved hydrophobic residues that was critical for the interaction with FliH. Thus, the interaction between FliI and FliH has similarities to the interaction between the N-terminal α-helix of the α-subunit and the globular domain of the δ-subunit of the F1-ATPase. This similarity suggests that FliH, by analogy with the δ-subunit of the F1-ATPase, may function as a molecular stator of the flagellum. The findings presented above have been published (96). The function of a putative H. pylori FliJ homologue, HP0256, was also investigated by knock-out mutagenesis. Disruption of this gene does not abolish flagellar assembly, however further research continued beyond this thesis showed that the knock-out mutant results in impaired motility.

As the Eucalyptus globulus plantation industry expands and matures in southwestern Australia (WA), the impact of disease within the plantation environment is predicted to increase. This thesis investigated the most abundant canker-causing pathogen associated with branch and stem cankers, Endothiella eucalypti the anamorph of Cryphonectria eucalypti. Endothiella eucalypti was widespread, although at low incidence, throughout the WA plantation estate and was frequently observed sporulating on the bark of healthy hosts in the absence of disease. Regions with a long (approximately 20 years) plantation history, such as Bunbury, had the highest incidence of this pathogen. A high degree of variability in pathogenicity, growth rate and colony morphology was observed between WA isolates of En. eucalypti. In the glasshouse, a significant variation in susceptibility of seven E. globulus provenances to En. eucalypti was observed. Although an interaction between the E. globulus provenance and En. eucalypti isolate was recorded, some provenances were generally more susceptible than others. In two 18-month-old plantations, the susceptibility of three provenances to En. eucalypti was significantly influenced by environmental conditions. Visual assessment of general tree health indicated that less healthy trees had smaller lesions than healthy trees. It is hypothesised that the selection of E. globulus provenances to suit site conditions in the future should decrease the risk of serious disease, especially on marginal sites. Endothiella eucalypti caused disease in intact stems of two-year-old E. globulus under glasshouse conditions. This suggests that En. eucalypti may not require a wound to infect in the field. Vegetative compatibility groupings between WA En. eucalypti isolates indicated a relatively high degree of genotypic diversity within the WA asexual population of En. eucalypti, whereas inter-simple sequence repeats PCR (ISSR-PCR) analysis indicated a lower level of genotypic diversity. Discrepancies between traditional and molecular techniques, such as ISSR-PCR, was attributed to the more specific gene-togene analysis afforded by molecular techniques. ISSR-PCR successfully distinguished variability within the En. eucalypti population and with the teleomorph, isolated in South Africa. It also separated Cryphonectria cubensis isolates from the C. eucalypti isolates. As copper is the micronutrient most limiting growth of E. globulus in WA, its role in the resistance of two E. globulus provenances was examined in a glasshouse trial. Lesion extension or defence responses of E. globulus to En. eucalypti did not differ between Cu-adequate and Cu-deficient plants. It is suggested that constitutive levels of host defence enzymes played a more important role in providing protection for the host against En. eucalypti than the external supply of copper. A reduction in the canopy volume of E. globulus within plantations due to insect herbivory or foliar pathogens, such as Mycosphaerella spp., has been reported to predispose the host to disease caused by non-aggressive canker-causing fungi. Under two separate glasshouse trials, conditions of 100% defoliation and 80% defoliation maintained over six weeks prior to inoculation, were required to significantly increase lesion extension caused by En. eucalypti in E. globulus stems. The ability of defoliated E. globulus to retain a degree of resistance to En. eucalypti was attributed to the rapid replacement of foliage and up-regulation of photosynthesis in remaining leaves. The carbohydrate reserves of the plant were depleted following defoliation and remained depressed regardless of the length of time the trees remained defoliated. In conclusion, the endophytic habit of En. eucalypti poses a threat to highly stressed trees, however it does not appear to be an immediate threat to WA plantation health. Although En. eucalypti has not yet been responsible for a major disease outbreak in WA, the impact of this disease on plantation-grown eucalypts elsewhere in Australia and worldwide serves as an indication of its potential to affect WA plantations.