The morfological and molecular identification of Fusarium verticillioides causing fusariosis on wheat grain

Abstract

During the 2014-2017 period, wheat samples were collected from discoloured spikes affected by Fusarium head blight (FHB) from 20 locations in Serbia. After isolation, fungi were cultivated on potato dextrose agar (PDA) at 25oC for 7 days. Based on the in situ identification on PDA, 36 isolates of the section Liseola were selected for further analyses. The pathogenicity of all isolates was confirmed on wheat leaves. The virulence of isolates was determined by measuring the lengths of spots formed at the inoculation leaf site. In order to prove the presence of the species Fusarium verticillioides, a pair of primers FV-F2/FV-R was used. This pair of primers amplifies the sequence of the gaoB gene, and it proved to be specific for the stated species. Moreover, for the same purpose, a pair of primers VER1-VER2 based on the calmodulin partial gene was used. The reference isolate RBG 1603 Q27 was used as a positive control. The pair of primers VER1-VER2 produced a band of the expected size - 578 bp in 18 isolates, while using FV-F2/FV-R, a 370bp long band confirmed the presence of F. verticillioides in 16 samples. Sixteen out of 18 isolates verified with VER1-VER2 were also identified as F. verticillioides with FV-FS/FV-R. No amplification was observed in a negative control.

Tokom perioda od 2014. do 2017. godine sakupljani su uzorci pšenice sa fuzarioznih klasova sa 20 lokacija u Srbiji. Nakon izolacije, gljive su gajene na KDA podlozi 7 dana u termostatu na temperaturi od 25oC. Na osnovu identifikacije in situ na KDA (krompir dekstrozni agar), 36 izolata sekcije Liseola odabrano je za dalje analize. Potvrđena je patogenost svih izolata na listovima pšenice. Virulentnost izolata utvrđena je merenjem dužina formirane pege na mestu inokulacije. Za dokazivanje prisustva vrste Fusarium verticillioides korišćen je par prajmera FV-F2/FV-R koji umnožava sekvence gaoB gena i koji se pokazao kao specifičan za navedenu vrstu. Takođe, sa istim ciljem korišćen je par prajmera VER1-VER2 dizajniran na osnovu dela genske sekvence za kalmodulin. Kao pozitivna kontrola korišćen je referentni izolat RBG 1603 Q27. Par prajmera VER1-VER2 obrazovao je traku očekivane veličine (578bp) kod 18 izolata, dok je pomoću para prajmera FV-F2/FV-R traka dužine 370bp potvrdila prisustvo F. verticillioides kod 16 izolata. Šesnaest od 18 izolata koji su identifikovani VER1-VER2 parom, takođe su identifikovani i FV-FS/FV-R prajmerima. U negativnoj kontroli nije bilo amplifikacije.