20.500.12556/RUL-138289 Vzpostavitev metode za ugotavljanje prisotnosti patogene glive Phyllosticta citricarpa Establishing a method for detection of a pathogenic fungus Phyllosticta citricarpa Karantenska patogena gliva Phyllosticta citricarpa je povzročiteljica črne pegavosti agrumov. Zaradi nevarnosti njene razširitve v evropski prostor v ogroženih nasadih potekajo vzorčenja zraka in dežja. Analiza vzorcev je potekala v sklopu magistrske naloge, kjer smo z uporabo optimiziranega postopka izolacije DNA in testov qPCR preverjali prisotnost glive. Poleg kontrolnih testov 18S (komercialni test, Applied Biosystems), Yeast (Hierro in sod., 2006) in FungiQuant (Liu in sod., 2012), za specifično določevanje glive uporabljamo testa PC (van Gent-Pelzer in sod., 2007) in PC-2 (Schirmacher in sod., 2019). Testa na podlagi zaporedja ITS ločujeta P. citricarpa od P. capitalensis in P. citriasiana, ne pa tudi od P. paracitricarpa, zato je bil naš namen vzpostaviti in validirati nov qPCR test TEF1, ki bi to omogočal. Z zastavljenimi poskusi smo dokazali njegovo strogo specifičnost za P. citricarpa, visoko občutljivost in robustnost. Pokazali smo linearnost testa v širokem območju in z večkratnim izvajanjem poskusov dokazali njegovo ponovljivost. Ocenili smo ga kot primernega za nadaljnjo uporabo v diagnostične namene. Analizirali smo realne vzorce zraka in ugotovili, da iskani patogen v njih ni prisoten, kar pomeni, da se še ni razširil v evropski prostor. The quarantine pathogenic fungus Phyllosticta citricarpa is the causative agent of citrus black spot disease. Due to the risk of its spread to the European region, air and rain sampling is taking place in endangered orchards. The samples were analysed as part of this master's thesis, in which the presence of the fungus is monitored using an optimized DNA extraction protocol and qPCR tests. In addition to the control tests – 18S (Applied Biosystems), Yeast (Hierro et al., 2006), and FungiQuant (Liu et al., 2012) – the tests PC (van Gent-Pelzer et al., 2007) and PC-2 (Schirmacher et al., 2019) are used for specific fungal identification. Tests based on the ITS sequence are used to distinguish P. citricarpa from P. capitalensis and P. citriasiana, but not from P. paracitricarpa. Therefore, our goal was to establish and validate a new qPCR assay TEF1 that would allow this. With the set experiments, we demonstrated its strict specificity for P. citricarpa, its high sensitivity and robustness. We demonstrated the linearity of the test over a wide range and proved its repeatability by repeated experiments. It was evaluated as suitable for further use in diagnostics. We have analysed real air samples and found that the target pathogen is not present in them, which means that it has not yet spread to the European region. Phyllosticta citricarpa črna pegavost agrumov patogena gliva rastlinski patogen izolacija DNA PCR v realnem času vzpostavitev metode validacija testa ITS TEF1 Phyllosticta citricarpa citrus black spot pathogenic fungi plant pathogen DNA isolation real-time PCR method establishing assay validation ITS TEF1 true true false [S. Fišer] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2022-07-14 07:15:43 2022-07-14 07:15:52 2022-09-15 04:16:30 0000-00-00 00:00:00 2022 0 Ljubljana 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577 204745 115677443 23209.pdf 23209.pdf 1 E5195497B9F7084E98C93772BB429DE0 79663ee554d21b66daacff11f52dd62c07a6e9f5fef541d79d1d55b03ccd5b56 cb5cbc4b-0333-11ed-8aca-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=158563 Biotehniška fakulteta 0 0 0