Molekularna identifikacija vrste Fusarium verticillioides patogena zrna strnih žita

Abstract

Gljive roda Fusarium spadaju u grupu ekonomski značajnih prouzrokovača fuzarioze klasa strnih žita. Vrsta Fusarium verticillioides je kosmopolitski rasprostranjena, ekonomski značajna i toksigena vrsta. Primarni domaćin ove vrste je kukuruz, ali je njeno prisustvo zabeleženo i na pšenici, durum pšenici i ječmu. Za potrebe izolacije DNK zasejano je 36 izolata Fusarium spp. koji su gajeni na KDA podlozi, 7 dana u termostatu pri temperaturi od 25°C. Molekularna identifikacija i karakterizacija izvršena je primenom prajmera specifičnih za vrstu F. verticillioides. Specifični prajmeri koji su korišćeni za identifikaciju su VER1/VER2, zasnovani na genu za kalmodulin, i FV-F2/FV-FR, zasnovani na gaoB genu. Odabranih 14 izolata je okarakterisano na osnovu tri regiona: ITS regiona primenom prajmera ITS1 i ITS4. ITS region je univerzalni region za identifikaciju gljiva. Zatim, izolati su okarakterisani i na osnovu delu gena za elongacioni faktor (TEF 1-α regiona) pomoću prajmera EF1/EF2. TEF 1-α region je primarni region za identifikaciju vrste F. verticillioides. Takođe identifikacija je izvršena i na osnovu delu gena za RNK polimerazu (RPB2) primenom 7cf/11ar prajmera. RPB2 region je sekundarni barkod marker za identifikaciju vrste F. verticillioides. Sekvenciranjem tri regiona dobijene su sekvence na osnovu kojih su rekonstruisana filogenetska stabla za svaki region posebno. Od 36 izolata, kod 14 izolata dobijeni su amplikoni očekivanih veličina sa prajmerima VER1/VER2 i FV-F2/FV-R. Identifikacija ovih izolata potvrđena je i na osnovu sva tri regiona (ITS, TEF1-α i RPB2).

Fungi of the genus Fusarium belong to the group of economically important species that cause fusariosis on small grain cereals. The species Fusarium verticillioides is a cosmopolitan, economically important and toxigenic species. The primary host of this species is maize, but its presence has also been detected on wheat, durum wheat and barley. For the purposes of DNA isolation, 36 isolates of Fusarium spp. were grown on PDA medium, for 7 days in a thermostat at a temperature of 25°C. Molecular identification and characterization was performed using primers specific for the species F. verticillioides. The specific primers used for identification were VER1/VER2, based on the calmodulin gene, and FV-F2/FV-FR, based on the gaoB gene. The selected 14 isolates were characterized based on three regions: ITS region using primers ITS1 and ITS4. The ITS region is a universal region for fungal identification. Then, the isolates were characterized based on part of the elongation factor gene (TEF 1-α region) using EF1/EF2 primers. The TEF 1-α region is the primary region for identification F. verticillioides. Identification was also performed based on part of the gene for RNA polymerase (RPB2) using the 7cf/11ar primer. The RPB2 region is a secondary barcode marker for species identification of F. verticillioides. By sequencing three regions, sequences were obtained on the basis of which phylogenetic trees were reconstructed for each region separately. Out of 36 isolates, 14 isolates obtained amplicons of expected sizes with primers VER1/VER2 and FV-F2/FV-R. The identification of these isolates was confirmed based on all three regions (ITS, TEF1-α and RPB2).