| Abstract | Bakteriozni palež ozbiljna je bolest biljaka iz potporodice Maloideae, posebno jabuke i kruške. Epifitocije su, iako sporadične, često pogubne ovisno o pojavi povoljnih klimatskih uvjeta, količini početnog inokuluma i virulenciji patogena, te osjetljivosti sorata domaćina. Ovisno o godini i sortama bakteriozni palež može biti poguban ili od sekundarnog značaja. Kod osjetljivih domaćina infekcija se širi tako brzo kroz stablo da se, jednom zaražena, stabla ne mogu spasiti, te odumiru u kratkom vremenu nakon prvog vizualnog znaka infekcije. Fitosanitarne mjere suzbijanja bolesti zahtijevaju osjetljive i pouzdane metode identifikacije. U slučaju bakterije Erwinia amylovora, uzročnika bakterijske paleži, postoje različite metode. Cilj ovog rada je usporedba osjetljivosti detekcije bakterije Erwinia amylovora putem seroloških i molekularnih metoda. Također, u radu je napravljena usporedba metoda izolacije DNA korištenjem komercijalnih kitova: DNeasy Plant Mini Kit i Mericon Food Kit. Rezultati su pokazali da je metodom DNeasy Plant Mini Kit dobivena veća koncentracija i čistoća DNA stoga se upravo ta metoda koristila za daljnje molekularne metode. U nastavku istraživanja uspoređivala se osjetljivost serološke metode LFD (Lateral flow immunoassays) korištenjem komercijalnih brzih seroloških testova: Pocket diagnostic Kit i AgriStrip diagnostic kit, gdje se kao osjetljivija metoda pokazao Pocket diagnostic Kit. Klasičnom PCR metodom uspoređena su dva različita protokola ispitujući također dva različita koncentrata reakcijske smjese master mixa (Promega i Takara). PCR protokoli korištenjem Takara matser mix su pokazali pouzdanije rezultate. Osim klasičnog PCR-a korišten je i Real time PCR prema tri protokola za tri različite ciljane regije (ITS, amsC i hpEa). Usporedbom svih dobivenih rezultata došlo se do zaključka da je PCR u realnom vremenu najosjetljivija i najpouzdanija metoda za detekciju bakterije Erwinia amylovora. |
| Abstract (english) | Fire blight is a serious disease of the plants of the subfamily Maloideae, especially apples and pears. Epidemics, although sporadic, are often fatal depending on the occurrence of favourable climatic conditions, the amount of initial inoculum and pathogen virulence, and the susceptibility of the host species. Therefore, at any site where fire blight is present, the disease can be devastating or of secondary importance, depending on the year and the varieties grown. In susceptible hosts, the infection spreads so rapidly through the tree that once infected, the trees cannot be rescued, and die shortly after the first sign of disease. Phytosanitary disease control measures require sensitive and reliable methods of identification. In the case of Erwinia amylovora, a causative agent of fire blight, several methods have been developed. The aim of this paper is to compare the sensitivity of detection of Erwinia amylovora by serological and molecular methods. Also, the paper compares DNA isolation methods using commercial kits: DNeasy Plant Mini Kit and Mericon Food Kit. The results showed that the DNeasy Plant Mini Kit method achieved a higher concentration of DNA, which was exactly the method used for further molecular methods testing. In the continuation of the study, the sensitivity of lateral flow immunoassays (LFD) belonging to serological methods was compared using commercial rapid serological tests: Pocket Diagnostic Kit and AgriStrip Diagnostic Kit was proved as more sensitive method. The classical PCR method compared through two different protocols and two different master mixes (Promega and Takara). PCR protocols using Takara matser mixes showed more plausible results. In addition to classic PCR, Real time PCR was also used according to three protocols using three different target regions (ITS, Amsc and hpEa). Comparing all the results obtained, it was concluded that real-time PCR is the most sensitive and reliable method for the detection of Erwinia amylovora bacteria. |
