20.500.12556/RUL-171717 Tarčno utišanje genov pri patogeni glivi Verticillium nonalfalfae z nanosom dsRNA in vitro Spray-induced gene silencing of specific genes in pathogenic fungi Verticillium nonalfalfae Gliva Verticillium nonalfalfae je patogena gliva iz rodu Verticillium sensu stricto, ki okužuje hmelj in povzroča bolezen imenovano verticilijska uvelost hmelja. Ker dosedanji ukrepi za zatiranje s fungicidi niso bili učinkoviti, smo želeli na dveh izolatih glive V. nonalfalfae, T2 in Rec, preizkusiti novo, inovativno metodo preprečevanja imenovano s pršenjem posredovano utišanje genov (»spray induced gene silencing«). Ideja temelji na podlagi znanja o mehanizmu RNA interference, ki v celici transkripcijsko ali post-transkripcijsko uravnava izražanje genov. Z nanosom dsRNA na rastlino bi tarčno utišali enega ali več pomembnih genov v patogeni glivi in tako povzročili njen propad ali oviranje rasti. V raziskavi smo tako v in vitro pogojih na micelij glive nanašali ustrezno količino tarčnih dsRNA, plošče z gojiščem inkubirali in v določenih časovnih točkah po tretiranju spremljali učinek dsRNA na rast micelija v primerjavi s kontrolo. Hkrati smo iz micelija ob vsaki časovni točki izolirali tudi celokupno RNA in z RT-qPCR metodo preverili raven izražanja tarčnih genov po tretiranju. Ugotovili smo, da ima metoda pri izolatu T2 večji potencial, saj je prišlo do večje inhibicije rasti in znižanja ravni izražanja genov kot pri izolatu Rec. Naše ugotovitve kažejo tudi na to, da bi bile za učinkovito zatiranje potrebno kontinuirno nanašanje dsRNA, ki bi bila z enkapsulacijo v nanodelce zaščitena pred zunanjimi vplivi. The fungus Verticillium nonalfalfae is a pathogenic species from the genus Verticillium sensu stricto, which infects hop plants and causes a disease known as Verticillium wilt. Since current control measures with fungicides have proven ineffective, we aimed to test an innovative control method called spray-induced gene silencing (SIGS) on two isolates of V. nonalfalfae, T2 and Rec. The idea is based on the mechanism of RNA interference, which regulates gene expression at the transcriptional or post-transcriptional level. By applying double-stranded RNA to the plant surface, one or more important fungal genes are silenced, thereby leading to the suppression of pathogen’s growth or even death of the pathogen. In our study, we applied a specific amount of target dsRNA onto fungal mycelia under in vitro conditions, incubated the culture plates, and at defined time points post-treatment, monitored the effect of dsRNA on mycelial growth compared to untreated controls. At each time point, we also extracted total RNA from the mycelia and used RT-qPCR to assess the expression levels of the target genes. Our results showed that the T2 isolate exhibited greater potential for gene silencing with this method, as indicated by stronger growth inhibition and a higher reduction in gene expression compared to the Rec isolate. Our findings also suggest that for effective control, continuous application of dsRNA will be necessary, ideally with encapsulation into nanoparticles to protect it from environmental degradation. Utišanje genov Verticillium nonalfalfae nanos dsRNA in vitro metoda Gene silencing Verticillium nonalfalfae dsRNA delivery in vitro method true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-08-31 07:17:23 2025-08-31 07:17:26 2025-09-17 03:50:48 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 261895 247315203 50785.pdf 50785.pdf 1 4E1CB6B5D41095F96C15F7E7D55A4733 6043f86c5a9c04622ee776f511a787dd2fb13568d1def0c75c3c60d3bf1c2168 a509f52d-8629-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=215099 Biotehniška fakulteta 0 0 0