• Aspergillus flavus
• aflatoxin
• AF-X1
• biological product
• maize

Candidato: Alessandra Civello
Relatore: Dott.ssa Susanna Pecchia
Correlatore: Prof. Andrea Serra
Corso di laurea: Biotecnologie Vegetali e Microbiche
Titolo: Reduction of toxigenic A. flavus infection and aflatoxin contamination in maize treated with biological product AF-X1

Corn (Zea Mays L.) is one of the most widely distributed and the third most important cereal crop species in the world (after wheat and rice). Maize is used as livestock feed, human food, biofuel and as raw material in industry: starch, oil, ethanol and food sweeteners. This crop is susceptible to several mycotoxigenic fungi and their presence is related to meteorological condition. In particular, Aspergillus flavus and Aspergillus parasiticus, fungi belonging to Aspergillus section Flavi, are the most relevant in safety issue because they are able to produce aflatoxins. Aflatoxins are secondary metabolites and are considered the strongest natural carcinogenic toxins by the International Agency for Research on Cancer (IARC). Several strategies have been studied and applied to reduce the risk of AFs contamination in maize and among them biological control has shown great promise. This strategy is based on the competitive exclusion of toxigenic by applying atoxigenic A. flavus strains in maize fields. In Italy, the biological product AF-X1 was authorized and applied in corn cultivation reserved to animal feed. The active ingredient of AF-X1 is an endemic non-aflatoxigenic strain of A. flavus selected for its ability to compete with aflatoxigenic strains reducing aflatoxins level in maize kernels. The aim of this work was evaluated the presence of toxigenic and atoxigenic A. flavus strains in corn fields treated and not treated with AF-X1. To evalue the efficacy of AF-X1(25 kg ha-1) were chosen two different locations in Tuscany: Fauglia (PI) and Capannori (LU). Each field was divided into treated and not treated plots where two types of hybrid (FAO 400 and FAO 600) corn were cultivated. The corn kernel analysis has provided for internal mycoflora of seeds previously surface sterilized with NaClO 1% (v/v). All Aspergillus spp. were isotaled on four different agar media: A. flavus and A. parasiticus agar (AFPA), Czapek Dox Agar (CZ), Yeast Extract Sucrose (YES) and Coconut agar medium (CAM). In this way, Aspergillus section Flavi, toxigenic and atoxigenic A. flavus isolates was selectively distinguished. Moreover, maize flour samples were analyzed for aflatoxins by high-performance liquid chromatography (HPLC) with fluorescent detection. As regards soil samples (T0 before and after T1 the treatment with AF-X1), serial dilutions were plated on AFPA modified by adding Triton X-100. Total and Aspergillus section Flavi colony-forming units (CFU) were counted and only these last were isolated in the same agar media (AFPA, CZ, YES and CAM) used for corn kernel analysis. Using the above methods, 499 and 265 Aspergillus section Flavi isolates from corn kernel and soil were analysed. In general, the corn kernel data have shown a considerable increase of atoxigenic A.flavus isolates in treated field. Between treated and not treated field, the mycoflora was considerable changed: Fusarium colonies decrease while atoxigenic A.flavus isolates increase (31.5-91.3%) in treated plots respect to the not treated one (1.7-41%). The aflatoxin B1 average concentration was 1.8 µg kg-1 and 9.5 µg kg-1 in maize flour from treated and not treated field respectively. The percent reduction in aflatoxin B1 concentration differed from 67.7% to 99.7%. The soil samples data has shown a decrease of toxigenic A.flavus strains in treated plots. At Capannori, 8 A.parasiticus strains were found in treated and not treated field.