2012 Annual Report
1a.Objectives (from AD-416):
1. Understand the phylogenetics of aflatoxin production through an examination of the population dynamics of aflatoxigenic and atoxigenic Aspergillus (A.) species in mixed cultures. Determine the potential for restoration of toxigenicity in toxigenic strains of A. flavus and development of toxicity in other species or genera which contain partial aflatoxin gene clusters.
2. Identify A. flavus pathogenicity factors required during invasion of oilseed crops and link these factors to aflatoxin contamination. Identify the critical enzymes that will allow the formulation of viable biological control strategies for a given crop situation.
1b.Approach (from AD-416):
Aflatoxins (AFs) are polyketide-derived, toxic and carcinogenic secondary metabolites produced mainly by Aspergillus (A.) flavus on cotton, corn, peanuts, and tree nuts. One of the main strategies for prevention of AF contamination in these crops involves introducing a non-aflatoxigenic competitor Aspergillus to the soil of the plants during the growing season. Recent work revealed that A. flavus has a complicated evolutionary history that includes a history of recombination. It is the purpose of this proposal to better understand A. flavus population dynamics in agricultural environments in order to more safely use biocontrol as a strategy to reduce crop contamination with AFs. To do this we will develop new competitor strains with improved properties for tracking their dispersal after introduction onto crops and with improved ability to over-winter in order to decrease the need for annual reapplication. Optimal candidates should be unable to produce the neurotoxin, cyclopiazonic acid, without altering their competitive ability. The potential for restoration of both AF- and CPA-producing ability of the atoxigenic strain in the laboratory or the field will be assessed. We will determine if the ability to produce hydrolases by the biocontrol strain is important for its competitive ability. With these studies, we expect to be able to either develop new biocontrol strains or improve the design of currently used biocontrol strains to reduce preharvest AF contamination. We also expect to provide additional insight into the evolution of diversity within A. flavus.
Six natural non-aflatoxigenic biocompetitor isolates (fungal strains) were made fluorescent by transforming with the gene for the jellyfish green-fluorescent protein (eGFP). Competition experiments to test the aggressiveness of eGFP-transformed biocontrol strains have been completed. To date tests have included competition between each non-transformed biocontrol strain and its GFP-transformed self, as well as between GFP-transformed biocontrol strains and toxin producers with unique chemically distinct profiles. Preliminary results of competition studies indicate that, for some biocontrol strains, the GFP-transformant appears more vigorous as both a spore producer (a seed-like structure produced by the fungus) and colonizer of cotton seed. Both the toxigenic strain and the GFP-transformed biocontrol strain had similar rates of growth and colonization potential. Mating tests were performed between each transformant (called MAT1-2) and a MAT1-1 toxigenic strain of Aspergillus (A.) flavus to determine if production of fruiting bodies and viable ascospores (fungal fruiting bodies) is possible. Ascospore formation was found to occur as early as two months after inoculation. From the six month crosses of single ascospore colonies, we found that not every ascospore inherited the GFP construct. For colonies that fluoresce some conidial chains were entirely fluorescent while others were entirely non-fluorescent and some conidial chains showed a mixture of fluorescent and non-fluorescent spores. Previously enzymes able to degrade fungal cell walls (hydrolases) found to aid in fungal invasion of plants. Xylanase, esterase and pectinolyase enzymes (with hydrolase activity against different components of the plant cell wall) were screened in several A. flavus non-aflatoxigenic isolates. Comparisons among the aflatoxin-producing and non-producing isolates revealed that only Afla-Guard (a commercially available biocontrol formulation of atoxigenic A. flavus) had a significantly reduced esterase activity, while no differences were found among the isolates for xylanase activity. To determine if xylanase is critical for A. flavus invasion of oilseed crops, A. flavus deletion mutants in one of the major xylanases (Xyl-) have been produced. The ability of this fungal strain to invade cotton bolls is being compared to isolates of A. flavus capable of xylanase production. By high-performance liquid chromatograhpy (HPLC) analysis we found that an enzyme that degrades starch (amylase) is significantly reduced in the veA mutant compared to the control strain in starch medium and also in corn medium. In addition, a reduction of protease activity (enzyme activity for breaking down in proteins) a mutant lacking the fungal developmental regulatory protein veA was also observed compared to A. flavus wild-type levels. A global analysis of secreted hydrolases produced by wild-type and the A. flavus 70 veA mutant found significant differences in the protein profiles and liquid chromatography/mass spectrometry being used to identify the proteins. The ability of these fungi to secrete hydrolases is also being studied following growth on peanut kernel extract broth.
Hydrolase production by non-aflatoxigenic and aflatoxigenic Aspergillus flavus. Biocontrol depends on the ability of the fungus to reach and penetrate the seed of the susceptible crop. An ARS scientist in the Food and Feed Safety Laboratory at Southern Regional Research Center, New Orleans, LA, in preliminary studies has found that among the hydrolytic enzymes necessary for seed invasion, esterase activity (an enzyme activity that breaks down specific organic compounds into acid and alcohol) is significantly reduced in the non-aflatoxigenic Aspergillus flavus biocontrol strain, Afla-Guard, compared to that of other strains considered for biocontrol and the aflatoxigenic strain. This result suggests that esterase activity is probably not critical for fungal colonization of plants susceptible to aflatoxin contamination.
Mating between non-aflatoxigenic and aflatoxigenic Aspergillus (A.) flavus. Non-aflatoxigenic strains of A. flavus are being used to reduce contamination of cotton seed, corn and peanut by aflatoxin-producing fungi. The success of this program depends on genetic recombination being a rare or non-existent event following field application of the biocontrol strain. An ARS scientist in the Food and Feed Safety Laboratory at Southern Regional Research Center, New Orleans, LA, found that, under optimized laboratory conditions, non-aflatoxigenic strains of A. flavus that are currently being used for biocontrol are capable of mating with aflatoxin–producing strains after only two months of incubation. The new strains arising from the mating produce conidial chains that are either entirely fluorescent, entirely non-fluorescent, or a mixture of fluorescent and non-fluorescent spores. These results clearly demonstrate that mating between soil populations and the biocontrol population is able to occur at least under these in vitro conditions.