Managing Aflatoxin Contamination of Louisiana Corn

INVESTIGATOR: Damann, K. E.; Sweany, R.; DeRobertis, C.

PERFORMING INSTITUTION:
Plant Pathology & Crop Physiology
Louisiana State University
Baton Rouge, Louisiana 70803

NON-TECHNICAL SUMMARY: Aflatoxin contamination of corn is a serious animal health and economic problem facing growers in the Gulf South. This project researches approaches designed to minimize the opportunity for or effects of aflatoxin contamination on the corn crop.

OBJECTIVES: Investigate biocontrol of aflatoxin in corn by identifying and using an aggressive atoxigenic isolate of Aspergillus flavus to compete with the native toxigenic mycoflora. Survey the A. flavus population from corn and soils and develop a fingerprinting technique capable of identifying individual isolates. Continue Liberty herbicide research for control of aflatoxin contamination and attempt to obtain a use permit. Determine the effects of chemical treatments using signal molecules involved in plant defense and plant hormones controlling growth and development on aflatoxin contamination. Investigate the post-harvest decontamination of aflatoxin in corn by chlorination.

APPROACH: Isolates obtained by plating kernels on AFPA medium from the Louisiana corn production areas and from soil will be screened for aflatoxin production using a plate cyclodextrin fluorescence assay using YCSD medium. Vegetative compatibility groups (VCGs) will be determined and DNA isolated for fingerprinting experiments. PCR-based fingerprinting protocols using rep-PCR primers, primers from an Aspergillus retrotransposon and RAPD primers will be investigated. A backup approach is to use RFLP analysis. Atoxigenic isolates from the survey will be screened in an in vitro kernel screening assay for ability to protect against aflatoxin contamination. Those showing promise will be compared in field experiments to a biocontrol isolate obtained from Georgia. Frequency of colonization of the kernels by the biocontrol isolate, confirmed by fingerprinting, will be one of the selection criteria for identifying the most effective isolate. Another will be the VCG distribution of the native mycoflora in the corn agroecosystem.

PROGRESS: 2004/01 TO 2004/12
The population biology study of Louisiana isolates of A. flavus was begun in the fall of 2003 from kernel samples submitted by county agents. Fifty-three isolates were recovered and, of these, only nine, or 17%, were shown to be toxigenic. The 2003 Ben Hur biocontrol experiment consisted of 16 200-foot rows treated with Afla-Guard, a proprietary atoxigenic A. flavus isolate whose conidia were applied to sterile barley seed as a nutritional substrate (Circle-One Global, Shellman, GA). The first question asked of this system was: Can the biocontrol isolate applied between the rows colonize the developing kernel in order to protect it from infection by the toxigenic isolates? Two hundred seed per row (3200 total) were plated on selective AFPA medium. One-half were infected with A. flavus. Of this half infected, 62% were VCG 24, presumably the Afla-Guard biocontrol fungus placed in the row. The other 38%, not VCG 24, were tested for toxigenicity using the ammonia vapor plate test. Again, only 17% were toxigenic. It seems that nature is already doing the biocontrol experiment: 83% of the corn isolates (non VCG24) were atoxigenic in two sets of data. The aflatoxin levels in the biocontrol treated and untreated plots were both low (<20ppb) and not different. The biocontrol fungus was shown to be capable of colonizing the developing kernels in the field. The 2003 Macon Ridge biocontrol experiment used the Afla-Guard fungus. In this case, only 179 of 1,600 kernels plated were infected with A. flavus (11% instead of the 50% at Ben Hur). On the positive side 79% (142) were VCG 24. Of the 37 not VCG 24, only three were toxigenic (8%). To be effective, the biocontrol isolate must be able to establish itself in the soil and be available to colonize subsequent crops without reapplication. To determine this, 42 soil isolates and 25 isolates from cobs left in the field were obtained in November after harvest and discing. The VCG of these isolates will be determined to see if they are the same as the Afla-Guard isolate originally applied (VCG 24, one not previously reported in Louisiana). The results of the 2004 Ben Hur biocontrol experiment indicated a much lower frequency (5%) of infection by A. flavus, only 158 of 3200 kernels plated; however, of these, 131 or 83% were VCG 24. Efforts are continuing to characterize the VCG of over 600 isolates from agents and the biocontrol experiments which were not VCG 24. We will also be characterizing the RFLP fingerprints of these isolates using pAF28 as a probe. To further aid characterization of the VCGs of our isolates, we have DNA isolated from VCGs 1-63, which should allow fingerprint identification of some of the unknown isolates to VCG without screening the unknown against the entire tester set. The native isolates, which are atoxigenic, also will be evaluated to determine their potential as biocontrol agents against toxigenic isolates. This will be done by employing the kernel screening assay. Co-inoculating the kernels in this in vitro test and determining which atoxigenic isolates provide the best minimization of toxin production will be candidates for use as biocontrol agents in the field.

IMPACT: 2004/01 TO 2004/12
The ability to isolate, determine toxigenicity, VCG and fingerprint Louisiana isolates of Aspergillus flavus from corn fields will provide a robust picture of the population biology of this fungus over time. This will allow assessment of biocontrol potential by atoxigenic isolates identified. The ability to determine VCG by RFLP fingerprints using the probe pAF28 and techniques originated by others will simplify the procedures required to place an isolate in a particular VCG. This approach should lead to effective sustainable biocontrol of aflatoxin contamination in the Louisiana corn agroecosystem. The research carried on in conjunction with Z-Y Chen is delving into the mechanisms of aflatoxin resistance by elucidating proteins associated with resistance. The role of the putative resistance proteins is being analyzed by using RNAi technology to interfere with the protein production and render a more susceptible phenotype, thus confirming their role in resistance.

PUBLICATIONS: 2004/01 TO 2004/12
1. Damann, K., Sweany, R., DeRobertis, C. 2004. Frequency of colonization of corn kernels by atoxigenic Aspergillus flavus applied as a potential biocontrol agent. Phytopathology 94(6):S23.
2. Chen, Z-Y, Brown, R. L., Cleveland, T. E., and Damann, K. E. 2004. Investigating the roles of an aflatoxin resistance-associated protein in maize using RNAi. Phytopatholgy 94(6):S17.
3. Chen,Z-Y, Brown, R. L., Damann, K. E., and Cleveland, T. E. 2004. Identification of a maize kernel stress-related protein and its effects on aflatoxin accumulation. Phytopathology 94:938-945.
4. Chen, Z-Y, Rajasekaran, K., Brown, R. L., Damann, K. E., and Cleveland, T. E. 2004. Removal of aflatoxin contamnination from food and feed crops. In: Jaiwal, P. K. (ed)Improving the Nutritional and Therapeutic Qualities of Plants (Metabolic Engineering and Molecular Pharming). Dordrecht: Kluwer Academic Publishers.

PROJECT CONTACT:
Name: Damann, K. E.
Phone: 225-578-1401
Fax: 225-578-1415
E-mail: kdamann@agcenter.lsu.edu

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