Characterization of Aspergillus flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis.
Midorikawa GE, Pinheiro MR, Vidigal BS, Arruda MC, Costa FF, Pappas GJ Jr, Ribeiro SG, Freire F, Miller RN
Aims: The aim of this study was to determine the genetic variability in Aspergillus flavus populations from Brazil nut and cashew and develop a polymerase chain reaction (PCR) detection method. Methods and Results: Chomatography analysis of 48 isolates identified 36 as aflatoxigenic (75%). One hundred and forty-one DNA bands were generated with 11 random amplified polymorphic DNA (RAPD) primers and analysed via unweighted pair group analysis; using arithmetic means (UPGMA). Isolates grouped according to host; with differentiation of those from A. occidentale also according to geographical origin. Aspergillus flavus-specific PCR primers ASPITSF2 and ASPITSR3 were designed from ribosomal DNA internal transcribed spacers (ITS 1 and 2); and an internal amplification control was developed; to prevent false negative results. Specificity to only A. flavus was confirmed against DNA from additional aspergilli and other fungi. Conclusions: RAPD-based characterization differentiated isolates according to plant host. The PCR primer pair developed showed specificity to A. flavus; with a detection limit of 10 fg. Significance and Impact of the Study: Genetic variability observed in A. flavus isolates from two Brazilian agroecosystems suggested reproductive isolation. The PCR detection method developed for A. flavus represents progress towards multiplex PCR detection of aflatoxigenic and nonaflatoxigenic strains in Hazard Analysis Critical Control Point systems.
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