Vesela T.: Influence of plant biological control on production of secondary metabolites of Papaver somniferum IV. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Kralove, Department of pharmaceutical botany, Hradec Kralove 2018. In the experimental part of the thesis was studied the influence of plant biological control on the production of the main pharmaceutically usable secondary metabolites of opium poppy (Papaver somniferum L.). The contents of alkaloids morphine, codeine, thebain, papaverine and narcotine were monitored in the capsules and stems. Two poppy varieties, Orbis and Lazur, were selected for the experiment. Half of the plots were treated with the fungicidal biopreparate Polyversum, containing the germinable spores of oomycete Pythium oligandrum. Pythium oligandrum directly attacks the fungal pathogens. It also uses indirect control mechanisms, induces resistance in plants and promotes their growth. The remaining untreated plots served as a reference check. The extract was prepared from the individual samples. Subsequent analysis was performed using high performance liquid chromatography. The results show no positive effect of treatment on the content of all quantified alkaloids in poppy straw. Biosynthesis of alkaloids and Pythium oligandrum activity can be...

Lot of attention is paid to biological control agents of plant pathogens as it will reduce the amount of pesticides used in agriculture. Pythium oligandrum oomycete is already used commercially in the form of watering and spraying. In this work the properties of other isolates were characterized, and the metabolic changes were studied in plants of oilseed rape (Brassica napus subsp. Oleifera), whose seeds were treated with preparates based on Pythium oligandrum. In the first part of the thesis the properties of compounds secreted by isolates of Pythium oligandrum were tested. The amount of phenolic substances and the activity of endoglycosidase endo-β-1,3-glucanase, cellulase, chitinase as well as proteases were measured to assess the ability of oomycete Pythium oligandrum mycoparasitically protect the plant. Substances, especially oligandrin, which during interaction with the plant are responsible for "priming", have been observed, enabling the plant to respond rapidly to pathogen infection through systemic plant resistance. Differences between individual isolates also manifested themselves in electrophoretic separation in the protein representation. In the second part of the work, the effect of rapeseed seed treatment with isolates of Pythium oligandrum on plant metabolism was monitored. While...

Kaman, P.: Influence of plant biological protection on production of secondary metabolites of Papaver somniferum III. Diploma thesis. Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2018, 77 pp. Diploma thesis was aimed to verify the impact of biological protection on the production of alkaloids of the opium poppy (Papaver somniferum, L.). Polyversum biofungicide was selected for this purpose, the active ingredient of which is oomycete Pythium oligandrum. The poppy tested were Orbis and Lazur high-morphine varieties. Extracts were prepared from the individual samples which were analyzed by HPLC. The following alkaloids were morphine, codeine, papaverine, noscapine and thebain. The average morphine content was around 0,5 %. The largest amount of 0,62 % was found in the poppyhead of untreated variant of the Orbis variety. Codeine for both varieties averaged 0,07 % in the poppyhead of untreated variant. Treated plants reached slightly lower values. The average papaverine content was around 0,01 % in the treated and also untreated variant, but only in poppyhead of the Lazur variety. Higher noscapine content was measured in the Lazur poppyheads, averaging 0,06 %. The content was only around 0,01 % for Orbis variety. The alkaloid content was...

Les recherches sur la lutte biologique (ou biocontrôle) par utilisation de micro-organismes connaissent un essor remarquable, les applications au champ étant cependant encore limitées en raison des variations d’efficacité dans la protection des plantes. Celles-ci sont souvent imputées à la non persistance des agents de biocontrôle dans la rhizosphère ou sur le végétal qu’ils sont censés protéger. Afin de réduire ce risque, une solution consiste à utiliser des micro-organismes isolés du végétal que l’on souhaite protéger. Dans le cadre de cette thèse, Pythium oligandrum, un oomycète colonisateur de la rhizosphère de nombreuses plantes dont la vigne, a été étudié pour lutter contre l’esca, une maladie du bois de la vigne pour laquelle il n’existe actuellement aucune méthode de lutte disponible. Des souches de P. oligandrum ont été isolées de la rhizosphère de ceps cultivés dans 3 régions viticoles (12 vignobles) du Bordelais présentant des sols variés : argilo-calcaire, sable-graveleux et graveleux. Les analyses des communautés fongiques et bactériennes obtenues par empreinte moléculaire (Single Strand Conformation Polymorphism) ont montré que, contrairement aux bactéries, les espèces fongiques différaient selon les régions. Des Pythium spp. aux oospores échinulées ont été isolées à partir des racines des ceps échantillonnés, avec une prédominance de P. oligandrum (séquençage de la région ITS). L’analyse des séquences des gènes codant pour le cytochrome oxydase I et une tubuline a permis de constituer 3 groupes de souches. Le séquençage d’autres gènes codant pour des protéines « élicitines-like » a indiqué que chaque souche présentait au moins un gène codant pour chacun des 2 types d’éliciteurs de P. oligandrum : l’oligandrine et les protéines de la paroi cellulaire (CWPs). Il apparaît que le type de sol et la microflore associée à la rhizosphère n’exerceraient pas une influence suffisante pour que la structure génétique des populations de P. oligandrum soient associées à un contexte tellurique particulier. En revanche, le type de porte-greffe et la méthode de désherbage (chimique ou mécanique) pourraient avoir une incidence sur la colonisation racinaire par P. oligandrum. Les relations entre P. oligandrum et les racines de la vigne ont été étudiées par analyse transcriptomique (microarray Vitis vinifera de 29 549 gènes). Les résultats obtenus montrent que de jeunes plants de vigne ont répondu à la colonisation racinaire par P. oligandrum en modifiant l’expression de gènes intervenant dans plusieurs voies métaboliques. Deux aspects a priori opposés ont été observés : P. oligandrum serait perçu comme (1) un agresseur contre lequel la plante a mis en place des réactions de défense mais en même temps, comme (2) un micro-organisme symbiotique car un certain nombre de modifications transcriptionnelles étaient similaires à celles reportées dans les interactions rhyzosphèriques symbiotiques (e.g. forte stimulation de gènes codant pour des subtilases). Un essai visant à induire chez la vigne une protection contre un champignon pathogène impliqué dans l’esca, Phaeomoniella chlamydospora, grâce à P. oligandrum, a été réalisé. La colonisation des racines par P. oligandrum a été associée à une réduction de la longueur des nécroses dues à P. chlamydospora. En adéquation avec ce résultat, l’analyse transcriptomique par RT-PCRq et microarrays a montré une surexpression de la voie de l’éthylène. Plusieurs gènes spécifiquement induits constitueraient des marqueurs de résistance qu’il conviendra de valider lors de prochaines expérimentations. / Biocontrol research based on the use of microorganisms is expanding very rapidly. However, the use of such bioncontrol agents is still too inconsistent to effectively protect plants in field applications. This phenomenon is often attributed to the non-persistence of biocontrol agents in the rhizosphere or on the plants. In order to reduce the risk of this happening, one solution consists in using microorganisms that are isolated from the plants needing protection. In this thesis, an oomycete called Pythium oligandrum, which colonizes the rhizosphere of many plants, including grapevine, was assessed for the control of esca, a grapevine trunk disease for which no control method is currently available. P. oligandrum strains have been isolated from the rhizosphere of vines cultivated in 3 wine-growing regions (12 grapevines) of Bordeaux with different types of soil: stony-sandy, silty and stony. Analyses of fungal and bacterial communities using a molecular fingerprinting method (Single Strand Conformation Polymorphism) showed that, unlike bacteria, the fungal species varied according to the sampling region. Roots of all the vines sampled were colonized by echinulated-oospore Pythium spp., with P. oligandrum strains predominating. Phylogenetic analyses based on the genes encoding the cytochrome oxidase I and one tubulin allowed these strains to be clustered into three groups. The sequencing of the elicitin-like genes, whose proteins are key components in inducing systemic resistance in plants, showed that each strain held at least one gene encoding for each of the two kinds of P. oligandrum elicitors (i.e. oligandrin and Cell Wall Proteins). Sequencing and molecular fingerprinting analyses showed thus that the type of soil and the rhizosphere microbiota did not shape the population structure of P. oligandrum. However, other factors such as the different kinds of rootstock and weeding management can also have an influence on the root colonization by P. oligandrum. The relationship between P. oligandrum and grapevine was studied using a transcriptomic approach (microarray Vitis vinifera, 29 549 genes). The results highlighted the modifications induced by young vines in response to P. oligandrum root colonization, in the genetic expression of several genes belonging to different metabolic pathways. Two aspects, that are usually opposed, were observed: P. oligandrum was perceived by the plant either (i) as a pathogen because certain defence reactions were triggered (e.g. calcium signalling, resistance genes, abscissic acid metabolism) or as (ii) a symbiotic microorganism since several transcriptional changes were similar to those reported in symbiotic interactions (e.g. induction of subtilase genes). An assay aimed at protecting grapevine against a pathogenic fungus involved in esca, and known to be responsible for wood necrosis, i.e. Phaeomoniella chlamydospora, was carried out. The root colonization by P. oligandrum was associated with a reduction in the length of necroses. In line with this result, transcriptomic analyses by microarrays and RT-qPCR showed overexpression of several genes, particularly those of the ethylene pathway. Some of these induced genes could be thus used as resistance markers, but this needs to be validated in further experiments.