Production of ochratoxin a and citrinin by Penicillium verrucosum and P. viridicatum from grain and herbal fodder collected in various regions of Russia

Over the past decades, significant progress has been made in elucidating the prevalence of fungi Penicillium subgenus Penicillium in the environment and their ability to produce a wide range of metabolites with toxic effects (T. Rundberget et al., 2004; J. O’Callaghanet al., 2013; M. Schmidt-Heydtet al., 2015). In a number of European countries, one of the species, the P. verrucosum Dierckx has been shown to predominate on cereals, and a direct relationship has been revealed between the grain infestation and the frequency of detection of nephrotoxins (F. Lund, J.C. Frisvad, 2003; M. Lindblad et al., 2004; S. Elmholt, P.H. Rasmussen, 2005). However, the available data on toxins produced by this fungus in grain products and feeds remain very limited (M.R. Bragulat et al., 2008; V. Koteswara Rao et al., 2011). In Russia, a series of publications has recently been devoted to the toxigenic potential of microscopic fungi affecting feed, including 11 species of Penicillium (A.A. Burkin et al., 2019; G.P. Kononenko et al., 2021). The whole set of cultural and morphological features previously accepted for the taxon P. viridicatum Westling (K.B. Raper et al., 1949), according to Russian researchers, turned out to be characteristic of isolates occurring with a frequency of 20 % in wheat, barley, oats, rye (E.A. Piryazeva, L.S. Malinovskaya, 2014) and with 1.3 % in dry grass feeds (hay, straw) (E.A. Piryazeva, 2017). Here, for the first time, it is shown that a set of isolates differing in terms of sites and years of collecting is mostly P. verrucosum , and its capability of toxigenic production in vitro is assessed. In addition, this work is the first in which P. verrucosum Dierckx and P. viridicatum Westling were discovered among isolates previously assigned to the taxon (according to K.B. Raper et al., 1949), and the characteristics of their toxin production in vitro were assessed. The aim of the study was to evaluate ochratoxin A (OA) and citrinin (CIT) production on grain substrates in a set of 33 isolates from grain and fodder sampled in various Russian regions in different years and deposited into a local collection as Penicillium viridicatum Westling. Prior to toxin production assessment, the modern morphological criteria were applied to check taxonomic assignment of the isolates tested. Species identification was performed in accordance with the guidelines (J.C. Frisvad, R.A. Samson, 2004). Cultures were grown on sucrose agar with yeast extract (YES), sucrose agar with creatine (CREA), Chapek agar with yeast autolysate extract (CYA) for 7 days at 25 °C and 30 °C. The acidity index was determined by the change in the CREA medium colour. Fungal inoculum was grown on Chapek-Dox agar for 7-10 days at 23-25 °C. After culturing fungi on moistened rice grain (7 days, 25 °C, without lighting), the OA and CIT content in biomass extracts was measured by indirect competitive enzyme immunoassay (ELISA) using certified commercial test systems (VNIIVSHE, Russia). According to the growth rate, colony pigmentation, acidity reaction, and the morphological traits when grown on a panel of 3 test media, 8 cultures were found to be P. viridicatum and 25 cultures were identified as P. verrucosum . None of P. viridicatum strains formed OA and/or CIT. All strains of P. verrucosum produced CIT and some of them OA. In 17 strains implementing the biosynthesis of both metabolites, the accumulation of CIT in the sample on average was about 15 times higher than OA. OA levels above 10 µg/g were detected in only 12 % of producers. The absolute majority (92 %) of P. verrucosum strains accumulated CIT in amounts of more than 10 µg/g, of which 48 % of 100 µg/g or more, which allows us to classify them as highly active producers. The data obtained suggest the involvement of the P. verrucosum species in co-contamination of OA and CIT of domestic grain products and herbal feeds.