Production of alternariol in the populations of grain feed-associated small spore Alternaria species

Modern science has strong evidence that Alternaria fungi pose a serious toxic hazard. Alternaria species can grow well on various substrates and in a wide range of temperatures and humidity, occupying different ecological niches in this way, and can produce several types of especially dangerous secondary metabolites (S.M. Tralamazza et al., 2018). The most well-studied Alternaria mycotoxin alternariol (AOH), a dibenzo-a-pyron derivative, exhibits high cytotoxicity, genotoxic and mutagenic effects (Z. Mao et al., 2014). However, the ability of Alternaria fungi to produce this toxin still remains poorly studied. In Russia, a significant prevalence of small spore Alternaria species on grain crops (Ph.B. Gannibal, 2004, 2006; T.Yu. Gagkaeva et al., 2012), and an increase in the frequency of Alternaria occurrence and AOH accumulation in grain and feed mixtures (G.P. Kononenko et al., 2019, 2020) have been recently reported. In this article, we first showed that the species A. tenuissima , A. alternata , and A. arborescens can cause AOH contamination of grain feeds. The work aims to investigate AOH production by the grain feed-associated Alternaria species. Alternaria fungi were isolated from 57 feed samples of different types (wheat, barley, corn and oats, sunflower seeds, wheat bran and mixed feeds). Monoconidial isolates identified by morpho-cultural features as A. tenuissima (Nees et T. Nees: Fries) Wiltshire, A. alternata (Fr.) Keissl, and A. arborescens E.G. Simmons, and another 14 isolates assigned to A. infectoria species group were cultured for 7 days at 25 °С on a panel of 4 mycological media, the potato-carrot agar (PCA), hay infusion agar (HAY), malt extract agar (MEA), and vegetable juice agar (V-8). AOH was detected in extracts using a certified commercial kit for indirect competitive enzyme-linked immunosorbent assay (ELISA). Among the isolates belonging to A. infectoria species group, 13 were devoid of producing ability while for one of them the accumulation of AOН was observed on all growth media in quantities 2.0±0.2, 14±3, 18±4 and 220±30 μg/g, respectively. Evaluation of the biosynthetic potential of A. tenuissima and A. alternata showed the highest degree of its realization on MEA growth medium in terms of the total number of producers and the share of highly active and superactive isolates. The total amount of AOH accumulation in these conditions for both species was almost the same and amounted to 73 and 71 μg/g, respectively. A. arborescens isolates provided the highest AOH production on V-8, HAY, and MEA media in amounts equal to 106, 64, and 31 μg/g, respectively. The peculiarities of metabolic response of A. tenuissima , A. alternata and A. arborescens species to environmental changes and a rapid method to assess toxigenicity of Alternaria fungi during taxonomic identification are discussed.