Diversity of Aspergillus flavus isolates from corn and soil in three different Tuscany areas treated with the biological product AF-X1

Corn (Zea mays) is a crop widely cultivated throughout the world and it constitutes a staple food in many regions. Corn is susceptible to several mycotoxigenic fungi and their presence is strictly related to meteorological conditions in the growing areas. In particular, corn is susceptible to some Aspergillus species belonging to the section Flavi like Aspergillus flavus that is able to produce aflatoxins particularly dangerous to both humans and animals. Aflatoxins are secondary metabolites produced by A. flavus and they have been classified by the International Agency for Research on Cancer (IARC) in the group I as human carcinogens. Different strategies have been developed to manage aflatoxins in crops and among them biological control has shown great promise. This strategy is based on the application of non-aflatoxigenic strains in maize fields to competitively exclude naturally aflatoxigenic strains in the same niche and compete for crop substrates. In Italy, the biological product, AF-X1, was authorized for corn cultivation destined to animal feed. The active ingredient of AF-X1 is a local non-aflatoxigenic strain of A. flavus selected due to its ability to compete with aflatoxigenic strains reducing aflatoxins level in maize kernels. In this research, 66 isolates of A. flavus collected from corn kernels and from soil in three different Tuscany areas, Fauglia (Pi) San Piero a Grado (Pi) and Capannori (Lu) treated and not treated with AF-X1 were analyzed. As internal controls a strain of A. flavus isolated from sorghum seeds of the biological product AF-X1 and two known aflatoxigenic isolates, 10768 and 10769, were included in the study. In order to evaluate A. flavus variability, phenotypic traits (aflatoxin-producing ability and sclerotia production) along with DNA polymorphism using the M13-ISSR minisatellite primer were used. All A. flavus isolates were characterized on four different media: PDA, CZ, YES and CAM to evaluate sclerotia production and to screen their ability to produce aflatoxins. Total genomic DNA was extracted from the 66 A. flavus isolates using a bead beating homogenization combined with a modified CTAB method and was then utilized for ISSR-PCR with the M13 minisatellite primer. PCR fingerprinting profiles were analyzed with the GelAnalyzer software and a binary matrix based on the presence (1) or absence (0) of bands, considering each isolate and the total number of minisatellite bands (20). The genetic similarities were calculated using the Jaccard similarity index and cluster analysis was performed with the UPGMA clustering algorithm. Molecular and phenotypic data obtained, along with field data (geographic areas, biological treatment), were also analyzed using the Gower similarity index. Among the 66 isolates of A. flavus examined 19 were toxigenic and 47 nontoxigenic. Moreover, isolates showed a great variability in sclerotia production (from 0 to over 50 per Petri plate). Molecular data showed a high variability among isolates: 95% of minisatellite bands between 410 and 1950 bp were polymorphic. There was no obvious clustering of isolates in relation to their geographic origin, source or aflatoxigenicity. Anyway, when the data were analyzed separately for each geographic location A. flavus isolates from corn kernels and from soil formed more obvious groups irrespective of aflatoxins production as described elsewhere in similar studies