Understanding Biological Control of Aflatoxin Contamination of Corn K. Damann, LSU; B. Bluhm, U of A; T. Allen, MSU Report to the National Corn Growers Association February 25, 2014 San Antonio, TX

Outline Introduction & rationale Field experiments Spore trapping results Hybrids for reducing aflatoxin contamination Future work

RATIONALE FOR UNDERSTANDING BIOLOGICAL CONTROL: Touching or physical interaction is necessary for intraspecific aflatoxin inhibition. There is specificity in the touch inhibition of aflatoxin synthesis, i.e. just because a nontoxigenic strain inhibits toxigenic strain A doesn’t mean that it can also inhibit toxigenic strain B. (PLoS ONE 6(8): e2347. doi.1371/journal.pone ) That means that each nontoxigenic biocontrol strain has a spectrum of toxigenic strains it can and cannot inhibit. In order to be able to protect corn from contamination a mixture of nontoxigenic strains which together have the ability to complement each other’s lack of inhibitory ability appears to be the best approach. There also appears to be specificity in which toxigenic strains in the soil can infect or not infect corn. (Phytopathology 101: ) Knowing the “infectious aggressive” strains in the soil (those which infect kernels) or characteristics of those strains gives us a target against which to select the most effective biocontrol strains. In order to determine the “infectious aggressive” strains we are spore trapping to determine which strains “fly” and when and how our biocontrol strains “fly”.

Suspended Disk Culture Screened ~ 50 tox - kernel isolates vs 53 tox +. Af 51 completely inhibited AFB1 of Af 53.

In well: atoxigenic isolate 51 + GS Medium Filter membrane In insert: toxigenic isolate 53 + GS Medium In well: ½ 53 + ½ 51 + GS Medium Filter membrane In insert: ½ 53 + ½ 51 + GS Medium Toxin No Toxin FILTER INSERT-PLATE WELL: Af 51 - /53 +

2010 St. Joseph Biocontrol Test

2013 FIELD EXPERIMENTS BARLEY INOCULUM BIOCONTROL Louisiana Mississippi Arkansas SPRAY INOCULUM BIOCONTROL Louisiana

Bluhm field test in Arkansas was flooded out and no data is available. Conclusion: Louisiana and Mississippi showed negligible contamination. The environmental conditions were not conducive to aflatoxin contamination

SPORE TRAPPING EXPERIMENTS

Ionic spore trap

Stub is vortexed in 300ul.001% SDS to remove attached conidia and liquid suspension spread on an AFPA selective medium plate. Colonies counted (>1000) and 10 / plate isolated and single spored resulting in 295 isolates over ~ 30 days. DNA extracted, quantified, and SSR PCR performed with selected primers. Gel patterns analyzed to determine the putative identity of the isolates. This was done in the Ben Hur fields where we knew the identity of the applied nontoxigenic biocontrol strains and toxigenic strain.

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2013/07 to 2013/08 Ionic Spore Trap Sampling from Ben Hur For each spore trap stub placed in a 15 ml tube, add 300 µl.001% SDS, vortex for 10 seconds and spread on a single AFPA plate. Try to isolate 10 colonies from each plate. W:/KDamann Lab/Cathy/Data/ to Ionic Spore Trap Sampling from Ben Hur Isolate # and Isolate Designation NumberDayDateTime # Hours Sampler Collected # Times Sampler Collected Total # of Colonies Detected Total # of Colonies IsolatedAverage Wind Direction ˚ Wednesday7/10/20133PM-9AM Thursday7/11/201310AM-8AM VCG4 3Friday7/12/20139AM-8AM VCG451VCG4VCG?VCG4 AGVCG4 4*Monday7/15/20139AM-8AM ,140,131VCG4 51 VCG4 5Tuesday7/16/20139AM-9AM VCG4 6Wednesday7/17/201310AM-7AM VCG4 51 7Thursday7/18/20138AM-9AM VCG4 8Friday7/19/201310AM-8AM AGVCG4 9Monday7/22/20139AM-10AM ,228,248VCG4AG VCG4 AGVCG4 10*Tuesday7/23/201311AM-8AM VCG?VCG4 51 VCG4 11Wednesday7/24/20139AM-7AM VCG4AGVCG4 AG 12Thursday7/25/20138AM-8AM VCG4 13Friday7/26/20139AM-8AM AG 51VCG4 AGVCG4 14*Monday7/29/20139AM-7AM 's*0259,0.5,286 15Tuesday7/30/20138AM-8AM VCG? 51 16Wednesday7/31/20139AM-8AM 's* Thursday8/1/20139AM-8AM VCG4 AGVCG4 18Friday8/2/20139AM-8AM VCG451 AGVCG4 VCG? 19Monday8/5/20139AM-8AM AG VCG4 20Tuesday8/6/20139AM-8AM VCG4AG51VCG4 21Wednesday8/7/20139AM-9AM VCG4 51VCG4AG 22Thursday8/8/201310AM-8AM VCG4 51 VCG4 23Friday8/9/20139AM-7AM AGVCG4 51 AG 24Monday8/12/20138AM-9AM ,154,168VCG451VCG4 25Tuesday8/13/201310AM-8AM VCG4 AG VCG4 26Wednesday8/14/20139AM-8AM VCG4 27Thursday8/15/20139AM-8AM AG VCG4 AG 28Friday8/16/20139AM-9AM VCG4 29Monday8/19/201310AM-9AM ,67,126VCG4 30Thursday8/22/201310AM-9AM ,141, Wednesday8/26/201310AM-8AM ,122,130,124VCG4AG 32Thursday8/29/20139AM-8AM ,81,247 AG 33Monday9/9/20139AM-9AM ,267,268,273,289,142, 120,138,132,128,158 VCG4 VCG?51 VCG451 34**Tuesday9/10/201310AM-9AM Total:

AG26/2958.8% VCG1 VCG4133/ % VCG80/2950.0% VCG?5/2951.7% /2957.5% Identified Isolates186/ %

HYBRIDS FOR REDUCING CONTAMINATION Agrisure 3000GT Agrisure Viptera 3111

FUTURE WORK Development of a high throughput industrial strength assay for aflatoxin production which can be used to screen for mutants affected in ability to “touch inhibit” or to be “touch inhibited”. Development of an Agrobacterium insertional mutagenesis and selection system for Aspergillus flavus to be used to generate mutants impaired in touch inhibition.

Harold Lambert A. flavus isolates in coconut cream with aflatoxin levels (ng/ml) (10µl 5x10⁵ spores/ml in 500µl coconut cream:H₂O incubated for 5 days at 30˚C) B13 D9 D10 D4 D3 D2 A13 E2 C19 C2 C3 C1 B19 B17 B9 B12 B5 B4 A17 A5 A6 A3 A2 A1 59,902 38,360 27,144 25, ,675 6,976 9,102 35, ,374 14,269 52,832 46, ,387 3,342 41, ,162 45,344 40,038 31,201

AG Af A flavus toxigenic 53 paired with biocontrol strains 10µl 5x10⁵ spores/ml in 500µl coconut cream:H₂O incubated 5 days at 30˚C.

USES OF THE “YELLOW” ASSAY Quick qualitative screen for aflatoxin production. Quick qualitative assay for detecting isolates having intraspecific aflatoxin inhibitory ability (touch inhibition) when paired (putative biocontrol strains). Quick assay for atoxigenic mutants impaired in intra- specific aflatoxin inhibition. (yellow when paired) Quick assay for toxigenic mutants impaired in ability to be inhibited. (yellow when paired)

Photo: Gary Payne, N. C. State Univ. Aspergillus flavus disease/ biocontrol cycle on corn Parasitic biocontrol = “competitive inclusion” or “competitive phenotype conversion” by “touch inhibition” or intraspecific aflatoxin inhibition by thigmo-down regulation of aflatoxin synthesis which is strain specific. Saprophytic biocontrol = “competitive exclusion” by lowering inoculum potential is epidemiology based. This provides sustainability.

QUESTIONS?