Toxin-producing small-spore Alternaria species from oat grain contaminated with alternariol

For many years, the problem of grain infestation with toxin-forming fungi Alternaria has been under the close attention (S.M. Tralamazza et al., 2018). Extensive studies have been carried out on wheat (M.T. Amatulli et al., 2013; M.E. Müller, U. Korn, 2013) and barley (V. Sanchis et al., 1993; T.T.T. Nguyen et al., 2018). The grain of oats has been studied much less, and it is still unclear which species of fungi of this genus and to what extent are responsible for the accumulation of the toxin alternariol (AOL). In our country, data have been obtained on the infection with Alternaria fungi of oat grains from a number of regions (O.P. Gavrilova et al., 2016; Yu.I. Vargach et al., 2019), as well as grain samples of varieties and lines from the VIR collection detected in field tests (A.S. Orina et al., 2017), however, the frequency of occurrence of the toxin was estimated for several regional lots in total (A.A. Burkin et al., 2015; G.P. Kononenko et al., 2020). In this study, it was established for the first time that the species A. tenuissima (Nees et T. Nees:Fries) Wiltshire, which is known as an active AOL producer, and to a lesser extent representatives of A. arborescens E.G. Simmons and the ‘ A. infectoria’ complex can participate in the contamination of oat grains. The aim of the work was to study the species affiliation and toxin-forming ability of Alternaria fungi isolated from oat grain with natural AOL contamination. The object of the study was a sample obtained in October 2020 from an agricultural enterprise of the Moscow Province (Odintsovo District) containing AOL in the amount of 630 ppb. Isolation of pure fungal cultures was carried out after surface sterilization of grain and its sowing on Chapek-Dox agar containing bile and antibiotics. Color, structure, and growth rate of colonies were described on yeast extract sucrose agar (YES) and potato-carrot (PCA) on day 8. To assess their toxin formation, oat grain and a panel of four mycological media were used - PCA, malt extract agar (MEA), hay infusion agar (HAY) and an analog of vegetable agar (V-8). After cultivation (7 days, 25 °C, without lighting) and extraction of biomass samples with a mixture of acetonitrile and water in a volume ratio of 84:16, AOL was determined by ELISA test (A.A. Burkin, G.P. Kononenko, 2011) with a detection limit of 0.01 μg/g. In the subepidermal mycobiota of the studied sample, representatives of the genus Alternaria were predominant, the degree of infection was 36.0 %, and they were accompanied by fungi Fusarium spp. (14.7 %) and Epicoccum spp. (2.7 %). After carrying out mycological procedures for the isolation and identification of Alternaria cultures, they were assigned to the species A. tenuissima (Nees et T. Nees:Fries) Wiltshire (7 strains), A. arborescens E.G. Simmons (2 strains) and to the species complex ‘ A. infectoria ' (4 isolates). On the grain substrate, all strains of A. tenuissima , A. arborescens and three isolates of ‘ A. infectoria ’ produced AOL in amounts of 370, 5 and 0.8 μg/g. When testing cultures under the same conditions on agar media, the intensity of AOL accumulation in A. tenuissima was highest on HAY and MEA (56 and 23 μg/g), in A. arborescens and ‘ A. infectoria ’ - on PCA and MEA. Taking this into account, commercial substrate MEA and oat grains are recommended for in vitro evaluation of the biosynthetic potential of fungi and their involvement in contamination of AOL grains in an expanded format. The cultural and morphological features of several cultures of A. arborescens and ‘ A. infectoria ’ and their ability to AOL biosynthesis are discussed in a comparative aspect.