In vitro degradation of diacetoxyscirpenol and T-2 toxin by use of Mucor racemosus fresen. f. racemosus isolate

Abstract

Under controlled in vitro conditions the capacity of the Mucor racemosus f. racemosus 1215/09 isolate to degrade type A trichothecenes (diacetoxyscirpenol - DAS and T-2 toxin) was observed in the liquid nutritive medium. According to previously performed experiments it was proved that the selected isolate, originating from sunflower meal, had the ability to degrade these fusariotoxins when growing on the modified Vogel's agar supplemented with crude extracts of DAS and T-2 toxin. In order to determine biodegradation of fusariotoxins, the liquid nutritive medium - SPY (5% sucrose + 0.1% peptone + 0.1% yeast extract, pH 6.2) was simultaneously inoculated with the isolate M. racemosus f. racemosus 1215/09 and: a) Fusarium semitectum SL-B (DAS producer) or b) F. sporotrichioides R-2301 (T-2 toxin producer). The SPY media, inoculated with single fungal isolates, were used as a control of toxin biosynthesis. The cultures were incubated at room temperature (21-26ºC) on the rotary shaker (175 rpm). After the 3-5-day incubation, the filtration of liquid cultures and the extraction of fusariotoxins from filtrates with ethyl-acetate were performed. Determinations of DAS and T-2 toxin were done by thin layer chromatography using silica gel G. Depending on the incubation duration, M. racemosus f. racemosus in the mixed culture with F. semitectum degraded from 90.0 to 99.97% of DAS present in the medium (40,000- 120,000 µg l-1), while in the mixed culture with F. sporotrichioides it degraded from 95.0 to 96.7% of T-2 toxin present in the medium (240,000 µg l-1). Sterile filtrates of mixed cultures and single culture of M. racemosus f. racemosus, obtained by passing liquid cultures through the 0.45-µm membrane filter and added to the SPY medium, did not affect degradation of type A trichothecenes that had been biosynthesised by isolates F. semitectum SL-B and F. sporotrichioides R-2301 in the liquid medium.

U kontrolisanim in vitro uslovima proučavana je sposobnost izolata Mucor racemosus f. racemosus 1215/09 da degraduje trihotecene tipa A (diacetoksiscirpenol - DAS i T-2 toksin) u tečnoj hranljivoj podlozi. Prethodnim eksperimentima je dokazano da odabrani izolat, poreklom sa suncokretove sačme, poseduje sposobnost razgradnje navedenih fuzariotoksina, koji su kao sirovi ekstrakti dodati u modifikovanu Vogelovu podlogu. U cilju utvrđivanja biodegradacije fuzariotoksina tečna hranljiva podloga SPK (5% saharoza + 0,1% pepton + 0,1% ekstrakt kvasca, pH 6,2) je zasejana u isto vreme izolatom M. racemosus f. racemosus 1215/09 i: a) Fusarium semitectum SL-B (proizvođač DAS-a) ili b) F. sporotrichioides R-2301 (proizvođač T-2 toksina). Kao kontrola biosinteze toksina korišćena je SPK podloga inokulisana pojedinačnim izolatima gljiva. Kulture su inkubirane na rotacionoj tresilici (175 o/min) tokom 3-5 dana na sobnoj temperaturi (21-26ºC). Nakon 3 do 5 dana inkubacije vršeno je filtriranje tečnih kultura i ekstrakcija fuzariotoksina iz filtrata etil-acetatom. Determinacija DAS-a i T-2 toksina je rađena tankoslojnom hromatografijom na silika gelu G. Zavisno od dužine inkubacije, M. racemosus f. racemosus je u združenoj kulturi sa F. semitectum degradovala 90,0-99,97% DAS-a prisutnog u podlozi (40.000-120.000 µg l-1), dok je u združenoj kulturi sa F. sporotrichioides razgradila 95,0-96,7% T-2 toksina prisutnog u podlozi (240.000 µg l-1). Sterilni filtrati mešanih kultura i pojedinačne kulture M. racemosus f. racemosus, dobijeni propuštanjem tečnih kultura kroz 0,45 µm membranski filter i dodati SPK podlozi, nisu uticali na razgradnju trihotecena tipa A koje su biosintetisali izolati F. semitectum SL-B i F. sporotrichioides R-2301 u tečnoj podlozi.