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Aflatoxin Reduction in Crops (ARC)
Aflatoxin Management through Improved Understanding of Aflatoxin-producing Fungi and the Aflatoxin Contamination Process
How do we know where the atoxigenic strain is?

We currently use two methods (Vegetative Compatibility Analysis and Pyrosequencing) to monitor the distribution of applied atoxigenic strains after application.

For Vegetative Compatibility Analyses, multiple isolates are collected from a single sample (either crop or soil) and membership of each isolate in the vegetative compatibility group (VCG) of the applied atoxigenic is determined.  To assign VCG, each isolate is mutated into a nitrate non-utilizing auxotroph and paired with tester mutants of the applied VCG.

This is the work flow for VCG analysis of a single crop or soil sample. 
At least two isolations are performed on each sample resulting in 12 to 15 isolates.  Each isolate is then stored in sterile water and subjected to a nitrate auxotroph selection process that involves two different chlorate containing media. 
Auxotrophs are also stored in sterile water and then paired with tester mutants (one niaD- and on cnx-).  Each sample requires at least 24 water vials and 66 Petri dishes filled with culture medium (if only 12 isolates are collected and everything works the first time).

Cotty, P. J. 1994. Influence of field application of an atoxigenic strain of Aspergillus flavus on the populations of A. flavus infecting cotton bolls and on aflatoxin content of cottonseed. Phytopathology 84:1270-1277.

Cotty, P. J. 1994. Comparison of Four Media for the Isolation of Aspergillus flavus group Fungi. Mycopathologia 125:157-162.
The second method we use is pyrosequencing which monitors the incidence of single nucleotide polymorphisms specific to the applied strain within pools of DNA representing the total A. flavus community.

Pyrosequencing uses a sequencing through synthesis method to quantify the frequency of single nucleotide polymorphisms (SNPs) at various loci within pools of DNA.  In our research, the DNA pool is derived from either environmental or crop samples. See the Pyrosequencing technology links at: http://www.pyrosequencing.com.

 

Pyrograms of DNA isolated from pure cultures of the atoxigenic strain AF36 and the high aflatoxin-producing S strain.  DNA is isolated from either soil or crop samples and the incidence of the two strains within the samples is determined by measuring the proportion of the DNA that has the strain specific single nucleotide polymorphisms.

Das M.K., Ehrlich, E.C., Cotty, P.J. 2008. Use of Pyrosequencing to quantify incidence of a specific Aspergillus flavus strain within complex fungal communities associated with commercial cotton crops. Phytopathology 98:282-288
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Agricultural Research Service		 ARC is housed at the University of Arizona,
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The University of Arizona, Department of Plant Sciences, Division of Plant Pathology and Microbiology, PO Box 210036, Tucson, AZ 85721
Current Updates by Gabriel Barker sargasso@email.arizona.edu Tel: (520) 626-5704 Fax: (520) 626-5944