Disruption of CTB2 gene in C. beticola isolates Ahlburg and Ferrara. A The 800 bp 5′ and 3′ fragments of CTB2 were amplified separately with primer pairs 1/2 and 3/4, fused with the hygromycin cassette by overlapping regions, and amplified with nested primers (including NotI and ApaI sites) before cloning into pGEMT. B Example of Southern blotting with a hygromycin probe, from left to right, wild type Ferrara (no band), Ferrara ectopic, Ferrara ctb2 gene disruption strains 2-1 and 2-2, wild type Ahlburg, Ahlburg ctb2 disruption strain 2-1, Ahlburg ectopic, Ahlburg ctb2 disruption strain 2-2 (band at 3.7 kb, black arrow). Dig VII marker (Roche) was used as DNA size ladder. C Confirmative PCR with CTB2 internal primers located adjacent to the integration locus (IntF and IntR), from left to right, wild type Ahlburg (band at 0.3 kb), Ferrara cbt2 disruption strain 2-1, Ahlburg disruption strain 2-1 (band at 2 kb) FΔctb2-2 and AΔctb2-2 produced the same PCR bands as the correspondent disruptants (data not shown).