Optimization of laboratory conditions for biosynthesis of type A trichothecenes

Abstract

Type A trichothecenes, T-2 toxin and diacetoxyscirpenol - DAS, belong to one of the most toxic groups of fusariotoxins. Although larger quantities of them can be found more often in cooler parts of Europe, regarding their metabolic characteristics and the types of illnesses they provoke, it is obvious that even smaller quantities of these toxins can cause serious health disturbances of humans and animals in climatic conditions of Serbia. Having in mind the importance of these substances, the aim of this study was to carry out the optimization of laboratory conditions under which screening of Fusarium spp. isolates from Serbia, regarding T-2 toxin and DAS production, should be done. Four cultures of Fusarium sporotrichioides originating from different regions throughout the world, were under present investigation: ITM-391 (Italy), KF-38/1 (Poland), M-1-1 (Japan) and R-2301 (Germany). According to the previous literature data, all of these isolates were T-2 toxin producers, and some of them were also DAS producers. The influence of medium composition (different C and N atoms sources microelements etc), as well as aeration (in liquid media), on biosynthesis process of these mycotoxins, in vitro conditions was investigated. In the case of most Fusarium sporotrichioides isolates, highest yields of T-2 toxin and DAS were achieved under the conditions of more intense aeration, and with the use of glucose (5 or 20%) as a C atom source. Fermentation in semi-synthetic liquid medium, using a rotary shaker, was more suitable for screening the toxicity of the fungal isolates in pure culture because of shorter period of incubation, more simpler sample preparation, obtaining less interfering materials in crude toxin extracts, and possibility for more precise definition of factors influencing the yield of trichothecenes.

Trihoteceni grupe A, T-2 toksin i diacetoksiscirpenol - DAS, predstavljaju jednu od najtoksičnijih grupa fuzariotoksina. Oni se u većim koncentracijama češće mogu naći u hladnijim regionima Evrope, ali, u skladu sa njihovim karakteristikama i vrstama oboljenja koja izazivaju, jasno je da i njihove manje količine mogu dovesti do ozbiljnih zdravstvenih poremećaja kod ljudi i životinja u klimatskim uslovima Srbije. S obzirom na značaj ovih jedinjenja cilj ovog istraživanja je bio da se izvrši optimizacija laboratorijskih uslova u kojima bi se ispitivala sposobnost za biosintezu T-2 toksina i DAS-a kod Fusarium izolata iz Srbije. Istraživanjem su bile obuhvaćene 4 kulture F. sporotrichioides poreklom iz različitih zemalja sveta: ITM-391 (Italija), KF-38/1 (Poljska), M-1-1 (Japan) i R-2301 (Nemačka), za koje je prethodno opisano u literaturi da su producenti T-2 toksina, a neke i DAS-a. Ispitan je uticaj sastava podloge (različiti izvori atoma ugljenika i azota mikroelementi i sl) kao i aeracije (u slučaju tečnih podloga) na proces biosinteze ovih mikotoksina in vitro uslovima. Kod većine izolata najveći prinosi T-2 toksina i DAS-a su dobijeni u uslovima veće aeracije i pri upotrebi glukoze (5 ili 20%) kao izvora ugljenikovog atoma. Fermentacija u tečnoj podlozi se pokazala kao pogodnija metoda za testiranje toksigenosti gljivičnih izolata od prirodnog sterilnog supstrata, zbog kraćeg perioda kultivacije, dobijanja sirovih ekstrakata toksina sa manje pratećih materija, kao i mogućnosti preciznijeg definisanja faktora koji utiču na prinos trihotecena.