Comparison of methods for determination of the toxigenic potential of Aspergillus parasiticus Sp. and Aspergillus flavus L. isolated from maize

Abstract

Maize is considered one of the most susceptible crops to mycotoxins worldwide. Compared to other mycotoxins, the greatest attention has been paid to aflatoxins, due to their potential carcinogenicity and due to significant and longstanding problems they can cause in humans and animals. A. flavus and A. parasiticus produce aflatoxins in many economically significant crops in both fields and storages. Because of the potential aflatoxin contamination of maize grain, the toxigenic potential of A. flavus and A. parasiticus isolates, originating from Serbia, was tested in the present study. Furthermore, various applied methods for detection of these mycotoxins were compared in the study. Cultural, serological and analytical methods for the detection of mycotoxins were compared in the course of the experiment by the direct extraction of aflatoxins from the nutrient medium. The cultural methods for the detection of aflatoxin production were applied to 20 isolates of A. flavus (MRIZP Af18-20) and A. parasiticus (MRIZP Ap1-17). These methods are based on the yellow pigment formation in mycelia and nutrition media, occurrence of fluorescence on PDA (potato dextrose agar), agar containing β-cyclodextrine (CD-PDA), as well as on the red pigment formation after adding ammonium hydroxide to the existing medium. The ELISA was used to check quantitative and qualitative analyses of total aflatoxins (B1, B2, G1, G2) while the HPLC method was applied to establish ability of isolates to synthesize aflatoxins B1, B2, G1, G2. The yellow pigment formation, fluorescence and colony color changes of isolates into red, as a proof of toxigenicity of isolates, were confirmed in all cases by ELISA. A high potential of total aflatoxin production was determined in the majority of observed isolates. The ability of A. parasiticus isolates to synthesize aflatoxins G1 and G2 was confirmed by the HPLC method. This was essential for a better understanding of the key role of the suitability of cultural methods for preliminary evaluation of a large number of isolates. Our goal was to employ rapid biochemical approaches to prevent aflatoxin contamination of crops, and to reduce human and animal exposure to foodborne mycotoxins.

Kukuruz se širom sveta smatra jednim od useva najpodložnijih za kontaminaciju mikotoksinima. Aflatoksinima se, u poređenju s drugim mikotoksinima, pridaje najveća pažnja zbog njihove potencijalne kancerogenosti, značajnih i dugoročnih problema koje izazivaju kod ljudi i životinja. Vrste A. flavus i A. parasiticus mogu produkovati aflatoksine kod mnogih ekonomski značajnih kultura u poljima i skladištima. Zbog potencijalne kontaminacije zrna kukuruza aflatoksinima u ovom radu je ispitan toksigeni potencijal izolata upravo ove dve vrste poreklom iz Srbije i upoređene su različite metode detekcije ovih mikotoksina. Tokom eksperimenta upoređene su odgajivačke, serološke i analitičke metode detekcije mikotoksina, direktnom ekstrakcijom aflatoksina iz hranljive podloge. Istraživanja su bazirana na primeni odgajivačke metode za određivanje produkcije aflatoksina kod 20 izolata A. parasiticus (MRIZP Ap1-17) i A. flavus (MRIZP Af18-20) poreklom iz Srbije. Odgajivačke metode su bile zasnovane na formiranju žutog pigmenta u miceliji i hranljivoj podlozi, na pojavi fluorescencije na PDA (krompir dekstrozni agar) i podlozi koja sadrzi β-ciklodekstrin (CD-PDA), kao i na obrazovanju crvenog pigmenta u podlozi nakon dodavanja amonijum hidroksida. ELISA test je korišćen za proveru kvantitativnih i kvalitativnih sadržaja ukupnih aflatoksina B1, B2, G1 i G 2, dok je HPLC metodom utvrđena koncentracija pojedinačnih aflatoksina B1, B2, G1 i G2. Obrazovanje žutog pigmenta, fluorescencija i promena boje kolonije izolata u crvenu, kao dokaz toksigenosti izolata, potvrđena je u svim slučajevima i ELISA testom. Kod većine izolata ustanovljen je visok potencijal produkcije ukupnih aflatoksina. HPLC metodom potvrđena je i sposobnost sinteze aflatoksina G1 i G2 od strane izolata A. parasiticus. Cilj eksperimenta bio je da se ispita efikasnost upotrebe brzih testova za detekciju aflatoksina, kako bi se sprečila kontaminacija useva i izloženost ljudi i životinja afla-toksinima.