Joe W' Dorner

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Development of Afla-guard for Control of
Aflatoxin in Peanuts
Joe W. Dorner
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Biological Control Concept Based on Competitive
Exclusion
Hypothesis Introduction of a dominant population
of a competitive, nontoxigenic strain of A.
flavus will lead to preferential invasion and
growth in peanuts by the introduced strain
resulting in reduced aflatoxin contamination.
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NPRL Environmental Control Plots
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NPRL Environmental Control Plots
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Afla-guard Development

• Proof of biological control concept

• Determine best isolate (strain) to use

• Determine application rate

• Formulation development

• Obtain EPA registration

• Determine efficacy in commercial use

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Proof of Concept
(1987)
Homogenized liquid cultures of a
non-aflatoxigenic, O-
methylsterigmatocystin (OMST)-producing strain of
A. parasiticus applied to peanuts early and
late-season.
Peanuts subjected to late-season drought stress.
Peanuts harvested and analyzed for aflatoxin and
OMST.
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Aflatoxin and OMST (ppb) in Peanuts
Untreated
Treated
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Determine Best Isolate (Strain) to Use
Aflatoxin (ppb)
Reduction
Isolate(s)
Year
Control
Treated
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Effect of Biocontrol Field Application on
Pre-and Post-Harvest Aflatoxin Contamination
Aflatoxin (ppb)
1999
1998
Pre
Post
Pre
Post
Treatment
Control
Af (NPL 45) Ap (21369)
Reduction
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Determine Best Isolate (Strain) to Use
Aflatoxin in Peanuts Treated with Nontoxigenic A.
flavus and A. parasiticus Separately and in
Combination
Isolate
Aflatoxin (ppb)a
Control
1635 a
A. flavus (NPL 45)
118 b
A. parasiticus (21369)
169 b
Af Ap mixture
127 b
a Means followed by the same letter are not
significantly different (P gt 0.05)
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Isolate Chosen for Registration
Aspergillus flavus NRRL 21882 (NPL 45)
Nontoxigenic no aflatoxin, CPA, or
aflatoxin-related metabolites
Naturally-occurring
Competitive
Stable Entire aflatoxin gene cluster deleted
vegetatively incompatible with toxigenic strains
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Determine Application Rate
Biocontrol Agents A. flavus and A. parasiticus
color mutants
(21368 and 21369)
Formulation Solid-state fermented rice
Aflatoxin Reduction with Different Application
Rates
Reduction
Rate (kg/ha)
1994
1995
22.4
78.1
74.4
89.6
95.0
112
560
90.2
99.9
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Formulation Development
Formulation Development
Formulations Used/Tested

• Aqueous conidial (spore) suspension

• Solid-state fermented rice

• Pesta extrusion product

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Pesta
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Formulation Development
Formulation Development
Formulations Used/Tested

• Aqueous conidial (spore) suspension

• Solid-state fermented rice

• Pesta extrusion product

• Corn flour granules

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Corn Flour Granules
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Formulation Development
Aflatoxin in Peanuts from Different Formulation
Treatments
Formulation
Aflatoxin (ppb)a
Control
405.5 a
SSF Rice
43.9 b
Pesta
20.4 b
Corn flour granules
29.9 b
a Means followed by the same letter are not
significantly different (P gt 0.05)
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Formulation Development
Formulation Development
Formulations Used/Tested

• Aqueous conidial (spore) suspension

• Solid-state fermented rice

• Pesta extrusion product

• Corn flour granules

• Compressed peanut hulls

• Spore-coated rice

• Spore-coated wheat/hulled barley

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Field Plot Formulation Testing
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Formulation Development
Aflatoxin in Peanuts from Different Formulation
Treatments
Formulation
Aflatoxin (ppb)a
Control
403 a
SSF Rice
43 b
Coated Rice
78 b
Coated Barley
89 b
a Means followed by the same letter are not
significantly different (P gt 0.05)
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Formulation Development
Spore-coated Hulled Barley
Afla-guard
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Afla-guard
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Formulation Development
Spore-coated Hulled Barley
Afla-guard
Economical
Rapid, large-scale production capability
Consistent
Stable
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Afla-guard Production Facility
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Obtain EPA Registration

• Oral toxicity of afla-guard to rats

• Honey bee field toxicity/pathogenicity test

• Acute intraperitoneal toxicity/pathogenicity (rat)

• Acute pulmonary toxicity/pathogenicity (rat)

• Acute respiratory pathogenicity (Bobwhite quail)

• Acute oral toxicity, infectivity, and
  pathogenicity (rat)

• All efficacy data

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Determine Efficacy in Commercial Use
Dooly Co. Georgia
Terrell Co. and Lee Co. Georgia
Geneva Co. Alabama
Clay Co. Georgia
Dale Co. Alabama
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Ground Application
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Aerial Application
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Afla-Guard on Soil Surface
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Sporulation of Nontoxigenic A. flavus
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A. flavus Soil Populations
(Preharvest)
CFU/g
Toxigenic
Treatment
Nontoxigenic


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Mean Aflatoxin (µg/kg) in Commercial Farmers
Stock Peanuts Treated and not Treated with
Afla-guard
Untreated
Treated
Location
Redn
P lt 0.01 P lt 0.001
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Farmers Stock Storage
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Aflatoxin in Shelled Stock Peanuts
(Warehoused in Unadilla, GA)
Untreated
Treated
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Aflatoxin in Shelled Stock Peanuts
(Warehoused in Dawson, GA)
Untreated
Treated
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Change in Peanut Value
Dawson
Unadilla
15 ppb
15 ppb
4 ppb
4 ppb


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Efficacy Study Summary

• Afla-guard reduced mean farmers stock aflatoxin
  by 85.2

• Afla-guard reduced the percentage of farmers
  stock lots with gt 100 ppb aflatoxin by 86.5

• Afla-guard reduced average shelled stock
  aflatoxin by 69 to 98