Bananas are an extremely important crop, serving as both a staple food in developing countries and as a dessert fruit in Western society. Two of the most devastating pathogens currently affecting both commercial and subsistence banana production are Fusarium oxysporum (Foc; causal agent of Fusarium wilt) and Mycosphaerella species (causal agent of black and yellow Sigatoka). Conventional breeding programs designed to improve the disease resistance characteristics of the commercially elite Cavendish cultivar have, thus far, been largely unsuccessful. Genetic engineering is now regarded as the most promising method to generate enhanced disease resistance in banana. In other crops and model species, strategies to enhance disease resistance have included the transgenic expression of defense-related genes such as; disease resistance genes (R genes), downstream signaling genes (eg. NPR1, non-pathogenesis related) and antimicrobial peptides (AMPs). The overall aims of this research were to amplify and compare the nucleotide binding site (NBS) domains of potential disease resistance genes from disease resistant and disease susceptible banana cultivars. To isolate and compare complete R gene sequences from these cultivars. To generate transgenic Lady Finger banana plants expressing the D4E1 antimicrobial peptide under the control of two different promoters and finally to assess extracts from these plants for their ability to inhibit the growth of Foc Race1. Using degenerate primers, the NBS domains of six resistance gene candidate (RGC) sequences were amplified from the disease resistant cultivar Calcutta 4 (C4) and the disease susceptible cultivar Cavendish (Cav). The RGC 1, 2, 5 and 6 sequences showed similarity to previously characterized R gene sequences isolated from monocotyledonous plant species, while RGCs 3 and 4 showed similarity to R genes which form part of the Fusarium wilt resistance locus isolated from the dicotyledonous species, Lycopersicon esculentum; as well as other monocotyledonous R genes. RGCs 1-4 and 6 were present and transcriptionally active in both C4 and Cav, whereas RGC-5 was present in Cav only and was not transcribed. The transcripts could not be detected by Northern analysis, which is consistent with previous reports that R genes are constitutively transcriptionally active at only low levels. The NBS domains of RGCs 1-6 showed less than 65% similarity (amino acid level) to one another but when each individual RGC isolated from the C4 and Cav gDNA and cDNA templates was compared the sequences showed greater than 97% similarity (amino acid level). Comparative sequence analysis revealed amino acid positions that were consistently different between the C4 and Cav clones. Southern analysis revealed that RGC 1-5 were present in both the C4 and Cav genomes in only low copy number (1-2 gene copies with 1-3 alleles), whereas RGC-6 showed high copy number in both cultivars. Complete RGC sequences were subsequently amplified by RNA-ligase-mediated (RLM) -RACE and 3'-RACE using specific primers designed to each of the RGC 1-4 NBS domains. Amplicons for each RGC were assembled to form potentially complete RGC sequences. Analysis of the sequences revealed the presence of coiled coil (CC) motifs in two of the amino terminal sequences while leucine rich repeats (LRRs) were identified at the carboxy terminal of all sequences. Multiple 3'-RACE products were amplified for each RGC sequence. Although the polyadenylated products were of different lengths, the sequences were greater than 98% identical at the amino acid level (except an RGC 3 clone which was 91-95% identical to the other RGC 3 clones due to a 37 amino acid deletion). Specific primers used to amplify each complete RGC sequence from both C4 and Cav DNA revealed that: RGC 1 (3.53 kbp) could be amplified from both C4 and Cav; RGCs 2 (2.99 kbp) and 4 (4.44 kbp) could be amplified from only Cav, however, the proposed truncations of these sequences (RGC 2: 1.3 kbp, RGC 4: 2.8 kbp and 2.9 kbp) could be amplified from both cultivars; RGC 3 (4.57 kbp) could not be amplified from either C4 or Cav, however, the three shorter sequences (1.96 kbp, 1.34 kbp and 1.28 kbp) could be amplified from both templates. The functional significance of the truncated sequences is currently unknown, however, truncated sequences have been detected in a number of R gene families isolated from other crops. No major sequence differences, such as deletions/insertions or early stop codons, were identified between the RGC sequences amplified from C4 as compared to Cav (greater than 91% amino acid similarity) and no sequence was identified as being present in the susceptible but absent from the resistant cultivar. However, comparative analysis of multiple clones isolated from C4 and Cav did reveal amino acid residues that were consistently different between the two cultivars. These differences may result in differing resistance capabilities, functional genomics studies would need to be undertaken to determine this. It has been proposed that CC-NBS-LRR type R genes employ NDR1/HIN1-like (NHL) proteins, after pathogen invasion is detected, in the signaling process that ultimately leads to the elaboration of a defense response. A NHL partial sequence (420 bp) was amplified from the C4 banana cultivar. The complete sequence of this gene (termed NHL-1) was isolated using RLM and 3'-RACE technologies (576 bp and 535 bp amplicons, respectively) and subsequently the 1.106 kbp sequence was PCR amplified from both the C4 and Cav cultivars. The banana NHL-1 gene contained conserved motifs/domains previously identified within other NHL-type gene sequences. These included a signal peptide motif, a transmembrane domain and three previously identified conserved motifs. Based on current research into NHL type genes, the banana NHL-1 sequence may not be useful as a transgene to enhance disease resistance in elite cultivars. However, it potentially plays an important role in the defense response signal transduction pathway and therefore will further our understanding of plant-pathogen interactions in banana. Transgenic Lady Finger banana plants expressing the D4E1 antimicrobial peptide under the control of either the maize polyubiquitin (Ubi) or banana bunchy top virus (BBTV) DNA-6 (Bt6.1) promoters were generated. These plants were subsequently assessed for the ability of their crude protein extracts to inhibit the germination of Fusarium oxysporum f.sp. cubense Race1 conidia in vitro. These anti-fungal bioassays revealed that fungal colony growth was reduced by 37-100% using extracts from the pUbi-D4E1 transgenic lines and 89-99% using extracts from the pBt6.1-D4E1 transgenic lines. The transgenic lines are currently undergoing multiplication in preparation for glasshouse and small plant challenge trials for resistance to Fusarium wilt. These preliminary results suggest that D4E1 may be useful in enhancing disease resistance in banana.

Marek’s disease (MD) is a highly infectious economically important oncogenic viral disease of chickens. It is found throughout the world and is caused by an alphaherpesvirus, Marek’s disease virus (MDV). Though this disease can currently be successfully controlled by vaccination, the virus has continuously evolved to greater virulence over the last several decades. Hence, there is a need for alternative approaches to control MD. Selection and breeding of MD-resistant chickens presents an attractive option for prevention of this disease. MHC-congenic chicken inbred lines, 61 and 72, which are highly resistant and susceptible to MD, respectively, have been identified, but the cellular and genetic basis for these phenotypes is unknown. The overall aim of this study was to investigate the cellular basis of resistance to MD using an in vitro MDV infection model with the hypothesis that resistance is exerted by the innate immune cells. MDV is a highly cell-associated virus which makes in vitro studies difficult. In vivo, MDV infects APCs (antigen-presenting cells: macrophages and/or dendritic cells [DCs]), B cells and activated T cells. Though both B and T cells can be infected in vitro, co-culture infection models have not been described for APCs. Thus, the primary goal was to develop a model for infecting these cells with MDV in vitro and to characterise infected and uninfected cells. Developmental studies used APCs derived from outbred chickens. Chicken bone marrow cells were cultured with chCSF-1 (for macrophages) or chIL-4 and chCSF-2 (for DCs) for 4 days and then infected by the addition of chicken embryo fibroblasts (CEFs) infected with recombinant MDV expressing GFP. CEF preparations naturally contain a mixture of CEFs (92-98%) and macrophages (2-8%) and both appear to be infectable with MDV. Infected CEFs were therefore separated from infected macrophages by FACS before adding to the bone marrow-derived APCs. Infected and uninfected APCs were sorted by FACS using GFP expression and APC-specific mAb staining (KUL01 and anti-CD45). Characteristic virus-infected and uninfected APCs were revealed via examination with live cell confocal microscopy. The presence of herpesvirus specific immediate early (ICP4), early (pp38), late (gB) transcripts and MDV specific transcript, L-Meq, in infected APCs was confirmed by RT-PCR providing evidence for MDV replication. Hence, a new in vitro MDV infection model of APCs has been established. Using the infected macrophages to infect CEFs showed that the infection was productive. This model was then extended to infect APCs of lines 61 and 72. Flow cytometric analysis revealed that a higher percentage of macrophages were infected in the susceptible line (72) than in the resistant line (61). To analyse this in detail, RNA-Seq was carried out to identify differentially expressed (DE) genes between the two lines pre- and post-MDV infection. From these DE genes, potential candidate genes involved in MD resistance and susceptibility were identified. Functional analysis of DE genes support the hypothesis that resistance to MD is determined at the macrophage level of the resistant line (61) and the JAK-STAT signalling pathway is at least one anti-viral mechanism by which this signature is expressed.

Master of Science / Department of Agronomy / William T. Schapaugh Jr / Sudden Death Syndrome (SDS) in soybean, (Glycine max ( L.) Merr.) caused by Fusarium virguliforme, is an increasing problem in commercial soybean production due to the yield loss associated with the disease. Screening for genetic resistance requires extensive visual evaluations. Canopy spectral reflectance may be an indirect tool for selection of SDS resistance as well as grain yield in large segregating populations. The objective of this study was to estimate SDS resistance and seed yield in large diverse soybean populations using canopy spectral reflectance. Spectral reflectance, disease index, maturity and yield were measured on two populations consisting of 160 nested association mapping recombinant inbred lines and checks; and 140 commercial cultivars with checks. Populations were grown in three environments in 2015 and 2016 with historic SDS disease pressure. Entry, environment, and entry by environment sources of variation were significant for disease index, yield, maturity and spectral reflectance. Changes in season average reflectance were correlated to disease index, yield and maturity. Estimation models of disease index, yield and maturity were created with season averages as well as individual day readings for both populations. Season average and individual day models accounted for 11% to 77% of the phenotypic variation in disease and 41% to 93% of yield variation when measurements were taken at the height of disease pressure. Models for disease index and yield models were able to predict significant portions of the phenotypic variation between entries at most environments. These results suggest that it may be possible to estimate resistance to SDS and grain yield in soybeans using spectral reflectance in breeding populations.

No description available.

Title: Analysis of self-medication with antibiotics in Kosovo Student: Ardita Veseli Supervisor: Prof. PharmDr. Jiří Vlček, Ph.D. Specialist Supervisor: Mgr. T. Belkina Department of Social and Clinical Pharmacy, Charles University in Prague, Faculty of Pharmacy in Hradec Králové Background: Self-medication is described as a behavior during which someone utilizes drugs for the purpose of treating self-diagnosed symptoms or disorders or the intermittent or continued use of a prescribed drug for chronic or recurrent disease or symptoms. When certain individuals self-medicate with antibiotics, they do it without any prior consultation with a physician. The antibiotics are bought from community pharmacies without a prescription, left-over antibiotics are used or they simply decide to follow the advice given from family members, neighbors or friends. Even though antibiotics are considered revolutionary therapeutic agents in order to eradicate microbes, the repeated and improper antibiotic consumption is contributing to the increased prevalence of antibiotic resistance which nowadays is one of the world's most pressing health problems. Aim: The main goal of the theoretical part is to describe self-medication with antibiotics, the patterns influencing it and some complications associated with this...

The incidence of infectious diseases in livestock is a major concern for animal breeders as well as for consumers. As a alternative approach to the use of prophylactic measures or therapeutic agents, infectious diseases can be contended by increasing the disease resistance of animals by genetic improvement. Animals can be selected either on a measure of their resistance (indicator trait) or on the presence or absence of some specific resistance genes in their genotype. A prerequisite to the latter approach is the identification of the genes, or QTL for quantitative trait loci, underlying the trait of interest. By means of sophisticated statistical tools, the QTL mapping strategy combines the information from genetic markers and phenotypic values to dissect quantitative traits into their individual genetic components. Some of the methodological aspects of this strategy are studied in the present thesis in the context of disease resistance in livestock. Indicator traits of the resistance (such as bacteria or parasites counts) are not always satisfying the normality assumption underlying most of the QTL mapping methods. In this context, the ability of statistical tests to identify the underlying genes (i.e. the statistical power) can be considerably reduced. We show that compared to the use of a non-parametric method, the use of the least-squares-based parametric method on mathematically transformed phenotypes gives always the best results. In the context of high number of ties (equal values) as observed when measuring resistance to bacterial or parasitic diseases, the non-parametric test is a good alternative to this approach, as far as midranks are used for ties instead of random ranks. The efficiency of QTL mapping methods can also be increased by use of simple combinations of repeated measurements of the same trait. As a result of analyses performed on real data sets in chicken and sheep, we show that much attention should be paid to obtaining good quality measurements, reflecting at best differences in terms of resistance between animals, before performing a QTL search. The appropriate choice of resistance traits as well as of the time of their measurement are, beside the choice of the method and the quality of marker information, among the most preponderant factors to guarantee satisfying results.

Anthracnose, caused by <i>Colletotrichum truncatum</i>, is a major disease of lentil (<i>Lens culinaris</i> Medik.) on the Canadian prairies. Resistance to the more virulent race Ct0 of the pathogen is extremely rare within the <i>L. culinaris</i> gene pool thus resistance is being introgressed from <i>Lens ervoides</i>. The overall hypothesis of this project was that resistance derived from <i>Lens ervoides</i> accession L-01-827A would provide uniquely low levels of resistance to <i>Colletotrichum truncatum</i>. Individual studies were performed to: i) test field-resistance of interspecific lines; ii) develop a cutting-method to generate replicated phenotypic data on single plants; iii) study the deterioration of resistance between the juvenile phase (JP) and adult phases (AP) in the line CDC Redberry; and iv) examine genetic control of resistance in a susceptible and partially resistant background. Field evaluations of <i>L. culinaris x L. ervoides</i> lines indicated resistance genes from the wild species will provide unprecedented levels of disease control and heritability of the trait was estimated to be moderate. Single plant phenotyping of resistance is unreliable, thus the feasibility of using clonal propagation of individual plants to generate replicated ratings was evaluated. Results showed consistency in segregation ratios between cutting and seedling-derived plants of the same population, thus the method was utilized for testing of genetic control of segregating populations. CDC Redberry showed varying disease levels depending on the age of the plants with resistance acquisition in the JP that decreased as the plants proceeded through the AP. The F₁, F₂ and F_2:3 generations of two introgression populations were tested for resistance. Resistance from <i>L. ervoides</i> to both races of <i>C. truncatum</i> appeared to be due to the same gene(s) or from the same linkage block in both populations. Models for genetic control were consistent between F₂ and F_2:3 generations, however were different between the populations depending on whether the interspecific line was crossed into a susceptible or partially resistant <i>L. culinaris</i> background. However duplicate recessive epistasis seemed to control susceptibility in the susceptible background and when JP resistance was not a factor. There were significant differences between the JP and AP on more than a third of the F₂s tested supporting different resistance gene action based on growth phase. Resistance in the JP seemed to be due to dominant and recessive epistasis. It was postulated that the the populations may have had segregation distortion commonly found in interspecific populations, thus the previously described genotypes for cotyledon colour and albino plants were used to test the hypothesis of segregation distortion. Segregation of these traits were found to be similarly distorted to previously reported interspecific <i>L. culinaris x L. ervoides</i> populations supporting the hypothesis that continued segregation distortion was found in the introgression populations. Overall, it was found that resistance derived from <i>L. ervoides</i> accession L-01-827A is a highly effective source for the lentil breeding program, however the result suggest more than one backcross to <i>L. culinaris</i> be necessary to properly integrate the resistance genes to eventually obtain fully fertile, adapted lentil cultivars.

Plants activate an array of defense mechanisms upon pathogen attack. Systemic acquired resistance (SAR) is an induced disease resistance phenomenon deployed after infection by a necrogenic pathogen and is dependent on endogenous accumulation of salicylic acid. The objectives of my research were to characterize SAR in the crop plant, <i>Brassica napus</i> (canola), and study the effects of overexpressing genes involved in SAR on disease resistance. Biological induction of SAR using necrogenic Pseudomonas syringae and chemical induction using benzo (1,2,3) thiadiazole-7-carbothionic acid reduced growth of the bacterial pathogen P. syringae and the fungal pathogen Leptosphaeria maculans. This growth reduction was associated with an increase in transcript levels of pathogenesis-related (PR) genes, one of the characteristic features of SAR. Transgenic plants expressing a bacterial salicylate hydroxylase gene (NahG), were more susceptible to the above pathogens and were delayed in accumulating PR gene transcripts, indicating a need for SA accumulation for SAR in B. napus. Expression of two SAR genes from Arabidopsis, DEFECTIVE IN INDUCED RESISTANCE 1 (DIR1) and NON EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), in <i>B. napus</i> enhanced resistance against virulent P. syringae without SAR pre-treatments. Putative orthologs of DIR1 and NPR1 (BnDIR1 and BnNPR1) were isolated from B. napus based on EST sequences. BnDIR1 and BnNPR1 display 71% and 66% amino acid sequence similarities, respectively, to the corresponding Arabidopsis proteins. Expression of BnNPR1 in Arabidopsis npr1 mutant backgrounds indicated that it was able to functionally complement these mutations. Expression of BnDIR1 enhanced disease resistance in both Arabidopsis wild-type and dir1-1 mutant backgrounds. Expression of DIR1, NPR1, BnDIR1 and BnNPR1, separately, in <i>B. napus</i> plants enhanced resistance against P. syringae. SAR pre-treatments further enhanced resistance of transgenic <i>B. napus</i> plants expressing DIR1 and BnDIR1 to <i>P. syringae</i>, indicating an additive effect. Expression of DIR1 in B. napus did not provide resistance against <i>L. maculans</i>. These results provide the first in-depth molecular characterization of SAR in B. napus, and in particular, provide new insight into DIR1 function not previously reported in Arabidopsis.

Plants activate an array of defense mechanisms upon pathogen attack. Systemic acquired resistance (SAR) is an induced disease resistance phenomenon deployed after infection by a necrogenic pathogen and is dependent on endogenous accumulation of salicylic acid. The objectives of my research were to characterize SAR in the crop plant, <i>Brassica napus</i> (canola), and study the effects of overexpressing genes involved in SAR on disease resistance. Biological induction of SAR using necrogenic Pseudomonas syringae and chemical induction using benzo (1,2,3) thiadiazole-7-carbothionic acid reduced growth of the bacterial pathogen P. syringae and the fungal pathogen Leptosphaeria maculans. This growth reduction was associated with an increase in transcript levels of pathogenesis-related (PR) genes, one of the characteristic features of SAR. Transgenic plants expressing a bacterial salicylate hydroxylase gene (NahG), were more susceptible to the above pathogens and were delayed in accumulating PR gene transcripts, indicating a need for SA accumulation for SAR in B. napus. Expression of two SAR genes from Arabidopsis, DEFECTIVE IN INDUCED RESISTANCE 1 (DIR1) and NON EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), in <i>B. napus</i> enhanced resistance against virulent P. syringae without SAR pre-treatments. Putative orthologs of DIR1 and NPR1 (BnDIR1 and BnNPR1) were isolated from B. napus based on EST sequences. BnDIR1 and BnNPR1 display 71% and 66% amino acid sequence similarities, respectively, to the corresponding Arabidopsis proteins. Expression of BnNPR1 in Arabidopsis npr1 mutant backgrounds indicated that it was able to functionally complement these mutations. Expression of BnDIR1 enhanced disease resistance in both Arabidopsis wild-type and dir1-1 mutant backgrounds. Expression of DIR1, NPR1, BnDIR1 and BnNPR1, separately, in <i>B. napus</i> plants enhanced resistance against P. syringae. SAR pre-treatments further enhanced resistance of transgenic <i>B. napus</i> plants expressing DIR1 and BnDIR1 to <i>P. syringae</i>, indicating an additive effect. Expression of DIR1 in B. napus did not provide resistance against <i>L. maculans</i>. These results provide the first in-depth molecular characterization of SAR in B. napus, and in particular, provide new insight into DIR1 function not previously reported in Arabidopsis.

Anthracnose, caused by <i>Colletotrichum truncatum</i>, is a major disease of lentil (<i>Lens culinaris</i> Medik.) on the Canadian prairies. Resistance to the more virulent race Ct0 of the pathogen is extremely rare within the <i>L. culinaris</i> gene pool thus resistance is being introgressed from <i>Lens ervoides</i>. The overall hypothesis of this project was that resistance derived from <i>Lens ervoides</i> accession L-01-827A would provide uniquely low levels of resistance to <i>Colletotrichum truncatum</i>. Individual studies were performed to: i) test field-resistance of interspecific lines; ii) develop a cutting-method to generate replicated phenotypic data on single plants; iii) study the deterioration of resistance between the juvenile phase (JP) and adult phases (AP) in the line CDC Redberry; and iv) examine genetic control of resistance in a susceptible and partially resistant background. Field evaluations of <i>L. culinaris x L. ervoides</i> lines indicated resistance genes from the wild species will provide unprecedented levels of disease control and heritability of the trait was estimated to be moderate. Single plant phenotyping of resistance is unreliable, thus the feasibility of using clonal propagation of individual plants to generate replicated ratings was evaluated. Results showed consistency in segregation ratios between cutting and seedling-derived plants of the same population, thus the method was utilized for testing of genetic control of segregating populations. CDC Redberry showed varying disease levels depending on the age of the plants with resistance acquisition in the JP that decreased as the plants proceeded through the AP. The F₁, F₂ and F_2:3 generations of two introgression populations were tested for resistance. Resistance from <i>L. ervoides</i> to both races of <i>C. truncatum</i> appeared to be due to the same gene(s) or from the same linkage block in both populations. Models for genetic control were consistent between F₂ and F_2:3 generations, however were different between the populations depending on whether the interspecific line was crossed into a susceptible or partially resistant <i>L. culinaris</i> background. However duplicate recessive epistasis seemed to control susceptibility in the susceptible background and when JP resistance was not a factor. There were significant differences between the JP and AP on more than a third of the F₂s tested supporting different resistance gene action based on growth phase. Resistance in the JP seemed to be due to dominant and recessive epistasis. It was postulated that the the populations may have had segregation distortion commonly found in interspecific populations, thus the previously described genotypes for cotyledon colour and albino plants were used to test the hypothesis of segregation distortion. Segregation of these traits were found to be similarly distorted to previously reported interspecific <i>L. culinaris x L. ervoides</i> populations supporting the hypothesis that continued segregation distortion was found in the introgression populations. Overall, it was found that resistance derived from <i>L. ervoides</i> accession L-01-827A is a highly effective source for the lentil breeding program, however the result suggest more than one backcross to <i>L. culinaris</i> be necessary to properly integrate the resistance genes to eventually obtain fully fertile, adapted lentil cultivars.