Multidisciplinary Approaches to Reduce the Impact of Corn Aflatoxin on Texas

Dr. Joseph Awika – Food TechnologyDr. David Baltensperger – Soil and Crop SciencesDr. Youjun Deng – Soil MineralologyDr. Joe Dixon – Soil MineralologyDr. Tim Herrman – Grain Science / State ChemistDr. Tom Isakeit – Plant PathologistDr. Mike Kolomiets – Molecular Plant PathologyDr. Kyung-Min Lee – Cereal ChemistryMr. Kerry Mayfield – Corn BreedingDr. Seth C. Murray – Corn BreedingDr. Gary Odvody – Plant Pathologist (Corpus Christi)Dr. Tim Phillips - Toxicology and Food ScienceDr. Wenwei Xu – Corn Breeding (Lubbock)

Funding: Texas AgriLife, Texas Corn Producers Board, USDA, USAID, NSF, many others.

- Highly toxic compound produced by fungus Aspergillus flavus

- Toxicity at low concentrations:- 20ppb action level / above 500ppb must be destroyed- Potent carcinogen

- Pre-harvest production in corn kernels- Can also occur on corn in storage

What is Aflatoxin?

Dr. Gary Odvody

Interaction BetweenPathogen, Environment and Plant

Aspergillus flavus spores

Suitable environment

Susceptible host: corn& others

Disease Triangle

Infection / resistance

Vs.aflatoxin

production

Kerry Mayfield

- Drought / heat stress- High temperature/ low soil moisture- Especially after silking – enter silks- Corn maturation into increasing heat/ stress

- Insects- Primarily Lepidotera- Stalk borers- Corn earworm (CEW), Fall armyworm (FAW)

- Wounding- Aflatoxin accumulation variable:

- Within an ear - maybe a few kernels- Across a field – drought hot spots, field edge

- Late planting increases stress

Environments Favorable for Aflatoxin

Dr. Gary Odvody

Corn Yields and Aflatoxin

Aflatoxin Loss Liability 2008

Office of the State Chemist Sampling 2008

Kerry Mayfield

Aflatoxin levels in corn 2009Office of the Texas State Chemist

* The color indicates the highest reported incidence in a county.

≤ 20 ppb50 – 300 ppb300 ppb500 ppb

Dr. Tim Herrman & Dr. Kyung-Min Lee

Plant Breeding – find andincorporate genes or traitsfor A. flavus resistanceinto hybrids.

Integrated Pest Management –identify agronomic practicesthat reduce A. flavus.

Plant Pathology – identifydisease factors, developcompeting atoxigenic strains.

Remediation – Find binders orprocessing methods to reducethe toxin or toxin absorbance. Food Science/ Veterinary

Nutrition – Identify processingmethods that can reduceaflatoxin.

Pre-Harvest Aflatoxin Contamination• Texas 2008 corn farm gate value = $1billion (13th in Country) • $14 million loss liability from mycotoxins in 2008• Most elite US germplasm is highly susceptible in Texas • Breeding is one of the least expensive and least intensive methods

for reducing aflatoxin contamination • No identified sources of complete resistance to Aspergillus / aflatoxin• Test in multiple environments with inoculation

Percent (%) Acreage with Aflatoxin Claims 2008

Aflatoxin Resistance Differs Across Corn Inbred Lines and Hybrids

Weslaco

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

1600.00

1800.00

2000.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Genotype

Afla

toxi

n (n

g g

-1)

Genotype Pedigree6 (Tx772/CML326)-B-B5-B-B/ Tester8 LAMA2002-14-B-B/ Tester

Mayfield et al, 2007

Kerry Mayfield

Corn Breeding Pyramid for Reduced Pre-Harvest Aflatoxin Contamination

- Husk Coverage - Long, cover tips- Tight and thick

- Maturity- Flowering time- Days to maturity

- Kernal hardness- Ear nod (ear droop)- Earworm resistance- Drought tolerance

YIELDRow

number Kernel

size

Ear length

LodgingKernel density

StressResistance Stay Green

How do we separate

these various correlated

traits ?

QTL Mapping - RIL Hybrids

WE Oil

CS Oil

CC Oil

CS Protein

CS Starch

CS Log AF

WE Log AF

WE Nodded Ears

135 RIL Testcrosses (B73o2 x CML 161)College Station , Weslaco and Corpus Christi3 Replications 15 QTL detected in traits

Kerry Mayfield

Tx714 FR2128 Tx772Tx714 FR2128 Tx772

Aflatoxin levels can be reduced by deleting a lipoxygenasegene LOX5 in corn under field conditions in different genetic

backgrounds – natural diversity / non-GMO

Dr. Mike Kolomiets

Corn ears: rep 5 showing relative yield and insect damage at Corpus Christi, TX in 2006. Conventional hybrids are shown by row and traits are shown in

columnsVT Pro Yieldgard CB Non-Bt

Hyb 3

Hyb 2

Hyb 1

Avg. 646 ppb Avg. 898 ppb Avg. 1220 ppb

Dr. Gary Odvody

Dr. Tom Isakeit

Bentonites as Adsorbents

• Economically feasible

• Socially acceptable

• Environmentally friendly

Ines Mulder Maria Guadalupe Tenorio Arvide

Dr. Youjun Deng & Dr. Joe Dixon

Testing Natural Bentonites for Adsorption Sites and Ability

41 samplesUSA (TX, WY, MS, ID), Australia, Brazil, Japan, Korea, Mexico, Mozambique, Spain, Turkey, South Africa

D-spacing & Adsorption Sites

Poultry test (on going)

Dr. Youjun Deng & Dr. Joe Dixon

Animal FeedingProtected animals from diet levels as high as 7,500 ppb.

Ghana

Human Supplement

Reduced urine levels of AFM1 after 3-months

intervention

Deliverable Product

Dr. Timothy Phillips

Dioctahedryl Smectite (clay-based enterosorption) therapies for aflatoxins and other food contaminants. - No adverse health effects from consumption of NovaSil clay

Sampling Procedures are Extremely Important and Limiting

- Very Expensive to measure ( $8 to $100+ / sample)

- Heterogenous within an ear, field, grain bin, shipping container

To make progress:

- Need increased testing speed, accuracy, and sample size while reducing costs

- In research, best inoculation procedures must be followed

HPLC-Tandem MS Method (Aflatoxin)- Fast, compared with the tradition HPLC-Fluorescence

method

- Specific, quantitative, sensitive (LOQ 0.5 ppb)110309-Diluted in 20% MeOH

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

%

0

100AF-STD-7 MRM of 10 Channels ES+

331 > 245 (AFG2)3.00e4

2.11

2.28

AF-STD-7 MRM of 10 Channels ES+ 329 > 243 (AFG1)

2.29e52.31

AF-STD-7 MRM of 10 Channels ES+ 315 > 259 (AFB2)

5.84e42.52

AF-STD-7 MRM of 10 Channels ES+ 313 > 241 (AFB1)

5.06e52.85

AF-STD-7 MRM of 10 Channels ES+ TIC

8.23e52.85

2.312.11 2.54

Multiple reaction monitoring (MRM) of Aflatoxin B1, B2, G1 and G2 in LC/MS/MS

Dr. Tim Herrman & Dr. Kyung-Min Lee

Increase Throughput of Testing With NIRS

• Aflatest® is expensive and time consuming• Need to screen 5,000-10,000 samples/ year• Near Infrared Reflectance Spectroscopy (NIRS)

www.foss.dk

Run samples on NIRS

Identify informative samples from spectra

Test 10% of samples for aflatoxin and/or

Aspergillus

Develop calibration equation

Use NIRS calibration equation to predict

contamination levels

• Equation built using non-AG with a red (3/4) and val (1/4) sets

• 2,5,5,1 SCVD both vis and NIR (Rsq = 0.8071)

• Plot predicted vs. Full non-AG

NIRS Results – Rsq. = 0.8071

Safety and Monitoring of Respiratory Aflatoxin and Particulates

Apex Lite Personal Air Pumps were used to sample ~700L of air. Two of eight samples had detectible levels of

respirable particulates (.1mg/m3 ; .28mg/m3)

Clearvue Cyclone Air Cleaning

http://www.texascorn.org/images/E0117201/AflatoxinCenter_final_web.pdf

Aflatoxin Center of Excellence of the

SouthCreating Linkages between:-States (Alabama, Georgia, Mississippi, North Carolina, Louisiana, Texas)

- Commodity Groups

- Public Research Institutions

- Research, Education, Extension

Plant Breeding – find andincorporate genes or traitsfor A. flavus resistanceinto hybrids.

Integrated Pest Management –identify agronomic practicesthat reduce A. flavus.

Plant Pathology – identifydisease factors, developcompeting atoxigenic strains.

Remediation – Find binders orprocessing methods to reducethe toxin or toxin absorbance. Food Science/ Veterinary

Nutrition – Identify processingmethods that can reduceaflatoxin.