Rhizomania, caused by beet necrotic yellow vein virus (BNYVV), has been considered as an economically important disease around the world because of the extreme reduction in sugar beet yield and sugar content in affected plants. The spread of rhizomania all over the world, including the emergence of resistance- breaking virus isolates, have been become a major concern for the plant pathologists and plant breeders aiming at improving sugar beet resistance to   BNYVV as well as better understanding sugar beet-virus interactions during disease development. The main focus of this project is to elucidate the role of P25-interacting partner, the VIP1 transcription factor, in activation of transcription.  The isolation of the gene-of-interest (VIP1) was performed by RT-PCR on total RNA preparations extracted from root tissue of sugar beet (Beta vulgaris ssp. vulgaris).  The isolated gene of interest was cloned using gateway system into a binary expression vector and the obtained construct was then transformed into Agrobacterium tumefaciens for analysing transient expression in the experimental host (Nicotinana benthamiana).  Dual-luciferase promoter activity assay was performed on isolated leaf discs co-expressing P25 and VIP1 and compared to appropriate controls.  Six promoter constructs were tested. However, we observed an increase in luciferase activity (1.8-4.2-fold) upon co-expression of P25 and VIP1 only for two constructs tested, although the increase was not supported by Student’s t-test at 0.05 significance level. Nevertheless, the luciferase activity assay data for these two constructs were consistent with RNA-seq and RT-qPCR data obtained previously showing upregulation of the expression of these two specific sugar beet genes during BNYVV infection in sugar beets.     Thus, the results support our hypothesis that the interaction of the virus virulence factor P25 with VIP1 transcription factor is needed to activate transcription of certain genes in the nucleus for the virus benefit.

L’amélioration de l’efficacité de l’utilisation de l’eau (WUE) s’avère un élément clé afin d’augmenter la durabilité des plantes cultivées lors de périodes de sécheresse. Dans ce contexte, cette thèse a étudié la relation entre la conductance du mésophylle au CO2 (gm), un élément contraignant l’assimilation en carbone, et l’hydraulique chez des peupliers hybrides, espèces à forte croissance et haute transpiration. Il a été démontré, à l’aide d’une méthode améliorée, que gm reste constante durant les premières étapes d’un stress hydrique jusqu’à un seuil de conductance stomatique (gs) d’environ 0,15 mol m−2 s−1. En diminuant expérimentalement les limitations causées par les stomates, gm et la conductance hydraulique des feuilles n’ont pas été altérées, suggérant une isolation hydraulique partielle du mésophylle. L’application d’acide abscissique (ABA) exogène et la cavitation du xylème ont mis en évidence le délai de réaction de gm par rapport à gs, suggérant également l’isolation hydraulique du mésophylle. L’isolation hydraulique serait en cause et entrainerait un délai dans l’acheminement de l’ABA, retardant ainsi la fermeture d’aquaporines et entrainant une déviation de l’eau vers la voie apoplastique et une diminution irréversible de gm. Toutefois, une diminution de la turgescence résultant de la cavitation entraine une diminution de gm réversible par pressurisation d’eau dans le xylème, suggérant un effet réversible au niveau de la membrane plasmique. Ces deux mécanismes peuvent également être observés lors d’un stress hydrique de plus longue durée. Ainsi, la diminution de gm est retardée de quelques jours par rapport à gs, suggérant un effet de l’ABA endogène. Toutefois, une variation diurne de gm après plus d’une semaine de stress suggère une réaction au changement journalier de turgescence. La variation de réponse de gs entre différents génotypes de peupliers et le délai de réaction de gm permettent d’augmenter le ratio gm/gs, et ce dernier est corrélé positivement à l’efficacité de transpiration, une composante foliaire de WUE. Ainsi, le ratio gm/gs serait un trait à favoriser dans l’amélioration de WUE afin d’augmenter la résilience des plantes cultivées à la sécheresse, surtout dans un contexte de changements climatiques où la fréquence et l’intensité des périodes de séchesse sont prédits d’augmenter. / Increasing water use efficiency (WUE) is a key objective to improve the sustainability of cultivated plants to drought. In that context, this thesis has studied the relation between leaf hydraulics and mesophyll conductance to CO2 (gm), a component that restricts carbon assimilation, in hybrid poplar clones, plants with high growth rates and transpiration. It was first shown, using an improved measurement method, that gm remained constant during the early stages of water stress and declined only below a stomatal conductance (gs) threshold of ~0.15 mol m−2 s−1. Removing stomatal limitations experimentally did not improve gm or leaf hydraulic conductance (Kleaf), which suggests a partial hydraulic isolation of the mesophyll. Furthermore, adding xylem-fed abscisic acid (ABA) or inducing xylem cavitation again showed a delayed gm response and hydraulic isolation of the mesophyll. Two mechanisms would be at play. First, in the case of ABA, hydraulic isolation would delay its delivery and eventually induce mesophyll aquaporins gating and a shift towards the apoplastic water pathway, thus decreasing gm. However, it is only with a decline in turgor caused by xylem cavitation that pressurizing water allows for a full restoration of gm, which suggests a reversible change at the plasma membrane level. Those two mechanisms can be observed during a longer water stress period. Thus, the delay in gm decline by a few days compared to gs suggests an ABA response and the partial hydraulic isolation of the mesophyll. After more than a week of water stress, diurnal changes in gm indicate a daily turgor variation. The variation in gs response to drought and the delay in the decline of gm increase the gm/gs ratio, which is positively correlated to transpiration efficiency, a foliar component of WUE. Thus, the gm/gs ratio was used to discriminate between five poplar genotypes, and the results suggest that it should be improved to increase WUE and the resilience of cultivated plants to drought, which is more than ever critical since drought events are predicted to increase in frequency and intensity under climate change.

Thesis (M. Tech. (Agriculture)) -- Central University of Technology, Free State, 2013 / ntroduction: Good quality Eucalyptus is of importance to South Africa’s pulp and paper industry. Limited land is available for forestry, therefore Eucalyptus with genotypes for good pulp and paper qualities, particularly hybrids, are bred and cloned via cuttings. Although these Eucalyptus clones keep the favourable genotypes in the population, many have difficulty with rooting. Research has shown that rhizobacteria can improve rooting. Thus, one strategy to enhance the rooting of cuttings is to use rhizobacterial preparations. The aim of this study was to characterise rhizobacterial communities of Eucalyptus hybrid and species and identify possible plant-growth promoting rhizobacteria (PGPR). Materials and methods: Rhizospheric samples were collected from Eucalyptus hybrids and species. The rhizobacterial communities were characterised using fatty acid methyl esters (FAME) analysis and denaturing gradient gel electrophoresis (DGGE). DGGE fragments were further sequenced to identify rhizobacteria. Results and discussion: FAME analysis successfully achieved a broad characterisation of the Eucalyptus hybrid and species rhizobacterial communities based on their fatty acid composition. Myristic acid (C14:0) was the most abundant fatty acid. DGGE profiles gave a molecular profile of the Eucalyptus hybrid and species rhizobacterial communities based on their DNA composition. Nitrosomona eutropha was present in all samples which illustrates a nitrogen-rich environment. Adhaenbacter aquaticus was unique to the better rooting Eucalyptus hybrid GU111. Conclusion: This study provided some insight into the diversity of rhizobacterial communities of Eucalyptus hybrids and species. Possible PGPR were identified and the observation made that the nature of the soil environment changes with the aging of the associated host. These findings allow further investigation into the formulation of potential rhizobacterial preparations for rooting enhancement of Eucalyptus cuttings.

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A Terra Preta Antropogênica (TPA) tem essa denominação porque é encontrada em sítios arqueológicos, onde viveram grupos pré-históricos e é considerada um dos solos mais férteis do mundo. Nela é encontrada grande quantidade de material deixado por grupos indígenas como fragmentos cerâmicos, artefatos líticos, e especialmente carvão pirogênico. Estudos realizados com o carvão pirogênico verificaram que ele aumenta a capacidade de trocas catiônicas nesses solos. Por meio de microscopia de fluorencência, foi observada a presença de microrganismos habitando esse carvão, no entanto, não se sabe quais seriam. Devido à falta de informações sobre a diversidade bacteriana nessas estruturas, este trabalho estudou a diversidade bacteriana em amostras de carvão pirogênico de solos TPA coletadas nos sítios Lagoa Balbina (Amazônia Central- Amazonas) e Mina I (Amazônia Oriental - Pará), através de técnicas moleculares independentes de cultivo. O estudo visou também comparar essa diversidade com a encontrada no solo de onde carvão foi isolado. As estruturas de carvão foram separadas fisicamente dos solos e seu DNA genômico total extraído e usado como molde em reação de PCR utilizando oligonucleotídeos do gene 16S rRNA para o Domínio Bacteria. O produto da PCR foi clonado em vetor e os clones foram sequenciados e comparados com o banco de dados de 16S rRNA do RDPX. Com a construção das bibliotecas de clones do gene 16S rRNA a partir das amostras de carvão pirogênico observou-se que existe maior número de bactérias desconhecidas no carvão pirogênico do que no solo onde ele foi isolado. Acidobacteria foi o filo predominante nas bibliotecas de carvão pirogênico das duas localidades de estudo, assim como na biblioteca do solo do sítio Mina I. Já na biblioteca do solo do sítio Lagoa Balbina houve predominância do filo Firmicutes. Por meio do método de rarefação foi possível constatar uma menor riqueza de UTOs nas comunidades bacterianas presentes nas estruturas de carvão pirogênico quando comparado à riqueza de UTOs das comunidades bacterianas cujo habitat é o solo. Mas quando se compara a riqueza de UTOs entre as estruturas de carvão isoladas das duas localidades, observa-se que a riqueza é maior no sítio Mina I. Os valores obtidos com os índices de diversidade revelaram menor diversidade de UTOs nas bibliotecas obtidas para o carvão pirogênico das duas regiões estudadas se comparado dos valores para as bibliotecas obtidas do solo da mesma região. Os valores obtidos com os métodos não paramétricos revelaram maior riqueza de UTOs para as bibliotecas do carvão do sítio Mina I e solo TPA do sítio Balbina. A análise da PCA revelou que as bibliotecas do sítio Balbina mostraram-se altamente similares. Em adição, a análise com S-Libshuff, verificou que todas as bibliotecas comparadas são significativamente diferentes quanto à composição das comunidades bacterianas. O carvão pirogênico não é uma estrutura inerte, pois é capaz de ser habitado por diferentes bactérias e a sua estrutura da comunidade bacteriana é diferente daquela de onde ele foi segregado / Anthropogenic Dark Earth (ADE) has this denomination because it is found at archeological sites, where prehistoric groups lived, and it is considered one of the most fertile soils of the world. In this soil a great amount of material left by indigenous groups was found as ceramic fragments, lithic workmanships, and especially pyrogenic black carbon. Studies accomplished with the pyrogenic black carbon verified that it increases the capacity of cationic changes in soils. Through fluorescence microscopy, the presence of microorganisms was observed inhabiting that black carbon, however, this community is still unknown,due to the lack of information about the bacterial diversity in those structures.This work studied the bacterial diversity in samples of pyrogenic black carbon of ADE soils, collected at the sites Lagoa Balbina (Central Amazon) and Mina I (Oriental Amazon), through molecular techniques independent of cultivation. The study also sought to compare that diversity with the one of the soil where black carbon was isolated. The structures of black carbon were separate physically from the soils and total genomic DNA was extracted and used as template in a PCR reaction, using primers of the 16S rRNA gene for the Bacteria Domain. The PCR product was used for construction of clone libraries and the clones were sequenced and compared with the 16S rRNA of RDPX database. The 16S rRNA gene clone libraries from the samples of pyrogenic black carbon, it shown that is a larger number of unknown bacteria in the black carbon than in the soil where it was isolated. Acidobacteria was the predominant phylum in the pyrogenic black carbon libraries from the both studied places, as well as in the soil library from Mina I site. However in the library Lagoa Balbina site there was predominance of the phylum Firmicutes. Through the rarefaction method it was possible to verify a smaller richness of OTUs in the bacterial communities presents in the pyrogenic black carbon structures when compared to the OTUs richness of the bacterial soil communities.But, when the OTUs richness is compared among the isolated structures of pyrogenic black carbon of the two places, it is observed that the richness is higher in the Mina I site. The values from diversity indexes revealed smaller diversity of OTUs in the pyrogenic black carbon libraries when compared with the soil libraries for the two studied areas. The obtained values with the nonparametric methods revealed larger OTUs richness in the black carbon library of Mina I site and in the ADE soil library of the Balbina site. The PCA analysis showed that the libraries of the site Balbina site were highly similar. In addition, the analysis with S-Libshuff verified that all of the compared libraries were significantly different in bacterial communities composition. The pyrogenic black carbon is not an inert structure, once it is capable of being inhabited by different bacteria, and its bacterial community structure is different from that one where is was segregated

Smooth manifolds have been always understood intuitively as spaces that are infinitesimally linear at each point, and thus infinitesimally affine when forgetting about the base point. The aim of this thesis is to develop a general theory of infinitesimal models of algebraic theories that provides us with a formalisation of these notions, and which is in accordance with the intuition when applied in the context of Synthetic Differential Geometry. This allows us to study well-known geometric structures and concepts from the viewpoint of infinitesimal geometric algebra. Infinitesimal models of algebraic theories generalise the notion of a model by allowing the operations of the theory to be interpreted as partial operations rather than total operations. The structures specifying the domains of definition are the infinitesimal structures. We study and compare two definitions of infinitesimal models: actions of a clone on infinitesimal structures and models of the infinitesimalisation of an algebraic theory in cartesian logic. The last construction can be extended to first-order theories, which allows us to define infinitesimally euclidean and projective spaces, in principle. As regards the category of infinitesimal models of an algebraic theory in a Grothendieck topos we prove that it is regular and locally presentable. Taking a Grothendieck topos as a base we study lifts of colimits along the forgetful functor with a focus on the properties of the category of infinitesimally affine spaces. We conclude with applications to Synthetic Differential Geometry. Firstly, with the help of syntactic categories we show that the formal dual of every smooth ring is an infinitesimally affine space with respect to an infinitesimal structure based on nil-square infinitesimals. This gives us a good supply of infinitesimally affine spaces in every well-adapted model of Synthetic Differential Geometry. In particular, it shows that every smooth manifold is infinitesimally affine and that every smooth map preserves this structure. In the second application we develop some basic theory of smooth loci and formal manifolds in naive Synthetic Differential Geometry using infinitesimal geometric algebra.

Red alder (Alnus rubra) is a tree species with high economic and ecological importance. It is subject to defoliation during unpredictable, episodic outbreaks of tent caterpillars (Malacosoma spp.) that result in reduced growth, decreased wood production, unsightly appearance and mortality in severe cases. Alder trees are weakened by severe and repeated tent caterpillar defoliation, and this can increase the susceptibility of the trees to other pests, diseases and drought. Repeated attack by tent caterpillars can cause decline in red alder populations, which can have potential negative impacts on the ecological and economic benefits of the species. Evidence from other species has shown that plants produce phytochemicals for defense against herbivores at a cost to growth and reproduction, but the relative magnitude of the cost of allocating available resources to defense depends on the level of the resources, and the plant genotype. The quality of a plant as food for herbivores is influenced by leaf physical and biochemical traits, and these traits change during a growing season or upon attack by herbivores. My research aimed to explore the defense mechanisms of red alder against western tent caterpillars (Malacosoma californicum) and determine the resistance variation among and within red alder populations, and to evaluate red alder available resource (nitrogen) allocation to defense and growth. Bioassay feeding trials were conducted in 2014 and 2015 with western tent caterpillars (WTC) (M. californicum) on twenty red alder clones from ten provenances. Phenology and quality of red alder leaves as food for the defoliators were analyzed to determine if budburst, leaf chemical content, water content or physical traits are major determinants of western tent caterpillars preference for red alder leaves. In another experiment, one-year-old seedlings from 100 half-sib red alder families were treated with two levels of ammonium nitrate (NH4NO3) in two growing seasons in a common garden. Growth, herbivore defense-related traits and root nodulation were measured and ranked among the plant genotypes and between the two nitrogen (N) treatments. Leaves from the two N treatments and different alder families were also used for bioassay feeding trials with WTC larvae to determine effects of N and genotype on red alder herbivory resistance. In my final experiments, I harvested and analyzed leaves from three-year-old red alder trees from five different families on eight dates from early April to mid-October 2016 to quantify oregonin and total phenolics concentrations, and wound induction experiments were conducted to determine if the concentrations of the chemicals vary during a growing season and upon attack by insects. Alder clones and families differed in percentage leaf area eaten by caterpillars and in leaf defense traits. The concentrations of foliar phenolic compounds negatively correlated with the percentage leaf area eaten by the caterpillars, but the results suggest a threshold, above which the concentration of each of the chemicals appeared to reduce WTC feeding, individually. Particularly, foliar oregonin concentration above 20 % leaf dry weight consistently appeared to reduce feeding by caterpillars. N availability had significant effects on red alder seedling total dry biomass and leaf N concentration. There was a clear trade-off between red alder seedling growth, and content of the phenolic compounds and leaf thickness, which supports the growth-differentiation balanced hypothesis in relation to resource availability. The concentration of oregonin varied during the growing season and there were no significant responses of any of the measured compounds to wounding. The results suggest that red alder foliar oregonin, condensed tannin and total phenolics are constitutive defenses and are not wound-induced. The effects of leaf total phenolic and condensed tannin concentrations on insect herbivory have been documented by past studies but the effects of oregonin concentration in red alder leaves on tent caterpillar feeding is a novel finding. / Graduate / 2020-04-18

Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications. The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line. Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency