Detection of Citrus tristeza virus in citrus trees by using modified ELISA method

Virus tristeza (Citrus tristeza virus, CTV) uzročnik je ekonomski značajne bolesti agruma. Ovisno o kombinaciji domaćina/genotipa virusa varira i intenzitet simptoma. U jake simptome ubraja se i jamičavost drva koju karakterizira deformacija drva inficirane biljke u obliku utora/jamica. Cilj rada bio je metodom ELISA provjeriti prisutnost virusa CTV u 46 indikatorskih biljaka koje su 2008. ciljano zaražene virusom te utvrditi eventualne razlike u „preživljenju“ različitih virusnih genotipova. Osim standardnom ELISA-detekcijom korištenjem homogenata floema, karakterističnog mjesta replikacije virusa, iskušana je detekcija virusa iz poprečnih prereza grana sa i bez pripadajućeg floema. Pretpostavka je bila da će modificirana metoda uz korištenje prereza grana s floemom dati rezultate identične onima dobivenim iz ekstrakata floema, dok će korištenje prereza grana bez floema dati pozitivne rezultate samo kod biljaka sa simptomima jamičavosti drva. Standardnom ELISA-detekcijom potvrđena je prisutnost virusa u 52,2% uzoraka te je uočena razlika u stopi „preživljenja“ različitih virusnih genotipova. Osjetljivost testiranja modificiranim postupkom korištenjem drva s floemom bila je nešto manja u odnosu na standardni ELISA-protokol. Modificirana metoda uz korištenje prereza drva bez floema potvrdila je prisutnost virusa u 66,7% uzoraka povezanih sa simptomima jamičavosti drva što ukazuje na potencijal modificirane metode u selektivnoj detekciji ovih virusnih genotipova.Citrus tristeza virus (CTV) is the causing agent of an economically significant citrus fruit disease. Depending on the combination of hosts/genotypes of the virus, the intensity of the symptoms may vary. Among strong symptoms is the stem pitting, characterized by a deformation of the wood of the infected plant characterized by the occurrence of small holes/pits. The goal of the work was to use the ELISA method to search for the presence of the CTV virus in 46 indicator plants, which were experimentaly infected in 2008, and to determine the possible differences in the “survival” of the different virus genotypes. Apart from the standard ELISA detection by using the phloem extracts, the characteristic tissue where virus replicates, detection of the virus from transverse cuts of the branches, with and without the phloem tissue was also conducted. The hypothesis was that the modified method when using the cuts with the phloem tissue will give results identical to those gained from the phloem extract, while using the branch cuts without the phloem will give positive results only in those plants in which the presense of stem pitting symptom has been observed. Using standard ELISA detection, the presence of the virus was confirmed in 52.2% of the samples, and a difference in the rate of “survival” of the different viral genotypes has been observed. The sensitivity of testing by the modified procedure using the wood with the phloem showed less sensitivity when compared with the standard ELISA protocol. The modified method using the cuts without the phloem confirmed the presence of the virus in 66,7% of samples related to stem pitting symptoms, which points to a prospective of a modified method in a selective detection of stem pitting causing viral genotypes