First Report of Fusarium verticillioides Causing Fusariosis on Triticale Grain in Serbia

Triticale (Triticosecale Wittmack) is a cereal crop species developed to combine positive traits of wheat and rye into a single plant (Arendt and Zannini, 2013). Due to its high protein content, favourable amino acid composition and nutritional values higher than maize, triticale is an excellent component for preparing feed for domestic animals (Đekić et al. 2019). In May 2017, discoloured spikes of triticale were observed in a field in Zemun Polje, Serbia, with a disease incidence between 15 and 20%. A total of 400 kernels obtained from discoloured spikes were surface sterilised with 1% sodium hypochlorite solution for 3 minutes and then rinsed 3 times with sterilized water and plated on potato dextrose agar (PDA) at 25°C incubated in the dark for 7 days. A total of 15 isolates of genus Fusarium on PDA formed cottony white to greyish purple colony with dark yellow to purple grey reverse and average colony. Obtained isolates were transferred to carnation leaf agar (CLA) and synthetic nutrition agar (SNA), for 7-day incubation in the dark at 25°C. Ten isolates formed "rabbit ear" monophialides which is the main characteristic of Fusarium verticillioides (Sacc.) Nirenberg. Microconidia are formed from monophialids in long chains. Their shape is oval with a flattened base, without septa (4.0-18.0 x 1.5-4.5 µm). Shape of macroconidia is slightly falcate to almost straight, (3.5-4.5 x 31-58 µm). Chlamydospores are not produced (Leslie and Summerell, 2006). The genomic DNA from one F. verticillioides isolate was extracted using the DNeasy Plant Mini kit (Qiagen, Hilden) according to the manufacturer's protocol. Molecular identification was confirmed by sequencing the internal transcribed spacer (ITS), translation elongation factor-1 alpha (EF-1α) and RPB2 (O'Donell, 2000). Sequence was deposited in GenBank as MZ664391, MZ666958 and MZ666957. This sequence was compared with the sequences of F. verticillioides strains registered in the GenBank database based on nucleotide similarity. It showed 100% identity to the sequences MT180471.1 (ITS), MN861767.1 (EF-1 α) and MT264836.1 (RPB2). Pathogenicity of all isolates was tested on 20 randomly selected triticale spikes in four replicates (Mesterházy et al. 1999). Inoculation was performed when half of the plants reached the flowering stage, by spraying with 20 ml of spore suspension (1 × 106 spores/ml from 7-day-old cultures on PDA). F. verticillioides RBG 1603 Q27 was used as a positive control. Inoculated spikes were covered with PVC bags for 48h. Discoloured spikes were observed after 3 week incubation in a greenhouse at 20°C with a 16h light/8h dark photoperiod. All inoculated spikes showed symptoms, similar to those from field infections. Control spikes were symptomless. The fungus was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. Based on morphological, molecular and pathogenic features the isolated fungus was identified as F. verticillioides. Fusarium species infect crop plants worldwide resulting in yield loss and reduced grain quality due to mycotoxin contamination (Asam et al, 2017). In Serbia F. verticillioides was isolated from wheat and barley grains (Stanković et al., 2012) and to the best of our knowledge, this is the first report of F. verticillioides on triticale grain in Serbia.