1. Breeding and Testing for Aflatoxin Resistant Corn Wenwei Xu Texas A&M AgriLife Research Lubbock, Texas we-xu@tamu.edu

2. Team Members PI: Wenwei Xu, Texas A&M AgriLife Research Co-PI: Seth Murray, Texas A&M University Co-PI: Gary Odvody, Texas A&M AgriLife Research, Corpus Christi Collaborators: Dewey Lee, University of Georgia Xinzhi Ni, USDA/ARS, Tifton, GA Brian Scully, USDA/ARS, Tifton, GA Matt Krakowsky, USDA/ARS, Raleigh, NC Hamed Abbas, USDA/ARS, Stoneville, MS Paul Williams, USDA/ARS, MS Mike Brewer, Texas A&M AgriLife Research, Corpus Christi, TX Tom Isakeit, Texas A&M University

3. Aflatoxins Aspergillus flavus Aflatoxin B1 (C17 H12 O6) Aspergillus flavus sporulating on corn

4. Aflatoxin contamination Produced by fungus Aspergillus flavus L. US federally regulated at 20 ppb – Above 500 ppb corn must be destroyed! Can lead to acute death –Kenya 2004: 125 people died, 192 poisoned (levels to 2,000ppb) –US 1998: 25 dogs died eating levels of 100-200 ppb for 3 months Potent chronic carcinogen, leads to stunting, and other health problems in humans and animals –1.7cm decrease in height for highly exposed children in Benin –Higher rates of liver cancer in China –Varying sensitivities among species Clean feed Aflatoxin contaminated feed Courtesy of Velazquez, Bailey, Deng, and Dixon; Texas AgriLife (2010 - unpublished data)

5. Pre-harvest aflatoxin contamination Texas 2008 corn farm gate value = $1billion (12 th in Country) $14 - $250 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 Percent (%) Acreage with Aflatoxin Claims 2008

6. Use recommended crop production practices:  Plant early.  Irrigate to reduce drought stress.  Harvest early.  Minimize insect damage.  Avoid kernel damage during harvest.  Dry and store corn properly - 13 % or less moisture.  Keep storage facilities clean. Approaches to Mitigate Aflatoxin Contamination

7. Use recommended crop production practices. Apply non-toxic A. flavus strains. Use clay as in the animal feed to bind aflatoxin. Develop and deploy aflatoxin resistant hybrids. Approaches to Mitigate Aflatoxin Contamination

8. Develop corn hybrids with low aflatoxin:  Use transgenic approaches to reduce the fungal growth or to block aflatoxin biosynthesis pathway.  Use the native resistance and improve adaptation to the southern environments. Genetic Approaches to Mitigate Aflatoxin Contamination

9. Develop new high yielding lines for making hybrids that can yield well and also have low aflatoxin in the southern states; Provide extensive testing of new inbred lines, hybrids, and technologies in the southern states for improving yield performance and reducing aflatoxin risks. Objectives

10. What we do to reach our goals?

11. Breeding Strategies For Aflatoxin Resistance Kernel resistance Kernel resistance Drought/heat tolerance CEW/FAW Resistance CEW/FAW Resistance Tight husk High yielding Low-aflatoxin Multiple-stress Tolerant hybrids High yielding Low-aflatoxin Multiple-stress Tolerant hybrids Coordinated testing Association mapping Association mapping Gene Expression New sources of resistance

12. Southeast Regional Aflatoxin Trials (SERAT) Initiated in 2004. Most members in this project are core members of the SERAT. Uniform testing in multiple locations in TX, MS, GA, and NC. In-kind support from the industry:  Monsanto  Pioneer  B-H Genetics

13. Harvesting yield trials Inoculating yield trials with colonized kernels Sporulatio n T. Isakeit Fourier Transformed Near Infrared Reflectance Spectroscopy FT-NIRS (Thermo Antaris II) Vicam AflaTest immunoaffinity columns

14. Silk channel inoculation: injecting A. flavus canidia

15. Aflatoxin Assay with VIACM Fluorometer Grind grain samples Mix ground samples with 80% methanol for 1 min Clean and filter extraction Column chromatography Measure with fluorometer

16. Where are we now in developing and deploying aflatoxin resistant corn hybrids?

17.  Inbred lines developed by Xu are used in commercial hybrids in southern U.S. since 2012.  This hybrid has high grain yield, excellent grain quality, and and low aflatoxin.  Inbred lines developed by Xu are used in commercial hybrids in southern U.S. since 2012.  This hybrid has high grain yield, excellent grain quality, and and low aflatoxin.

18. Teosinte as a Potential Source of Resistance? Ears of BC 3 F 1 plants with 6.25% teosinte germplasm.  Selfed ears of F 1 plants  Teo-Lines

19. Disease Resistance and Yield of Teo-line Testcrosses HybridsRustAflatoxin (ppb)Grain yield (bu/a) WS MSETHFHLCSWSMean Teo-Line20 x NS11.0237155224024484151145173 Teo-Line29 x NS11.5216300235231114224145190 Teo-Line31 x NS11.05199237206100182137172 Teo-Line33 x NS11.0505559215232111187134176 Teo-Line48 x NS15.54309207237110180147176 Teo-Line49 x NS11.50564214227106157135168 Teo-Line54 x NS11.51475241243125171 190 Teo-Line62 x NS11.5191002245250115176154188 Teo-Line63 x NS11.0260697236238116148163180 DKC66-235.0157196724626998159149184 DKC67-874.0160860254274123173165198 P31G963.073216258268109182160195 P33D493.52813752261241105187156190 Test mean (50)2.4166698225238103172143176 CK mean3.91681699255263109176157192 CV%43.78813119 LSD 0.052.138 283625

20. All with brown midrib (BMR) trait. Non-stiff stalk group. Excellent silage yield. Excellent grain yield in central Texas. Texas A&M AgriLife Research recently released five new stress-tolerant corn inbred lines: Tx206, Tx207, Tx208, Tx209, Tx210 Texas A&M AgriLife Research recently released five new stress-tolerant corn inbred lines: Tx206, Tx207, Tx208, Tx209, Tx210

21. Inbed lines: FAW 1382 and FAW 1430  Resistant to FAW.  Low in aflatoxin. Dr. Xinzhi Ni, USDA-ARS, Tifton, GA

22.  Released inbred lines.  New lines in the pipeline.  Made two new synthetic populations with most resistant lines. Dr. Seth Murray, Texas A&M University

23. Four lines have now been released by Texas AgriLife research for improved aflatoxin control “REDUCED AFLATOXIN BETWEEN 30 % AND 73% REDUCTION…” Mayfield et al. 2012 Journal of Plant Registrations 6:88–94

24. Pedigree Bushels / Acre Aspergillus rating LAMA2002-58-3-B-B-B-B-B-B-1-B19A 72A 0.005 DK888 N11 F1S3 2141 2 34-B-2-1AB 61A 0.010 (NC300 x Tx714-B/B104-1/CML343)-2-1-B-B-B-B-B-B-B-B-1-B25AB 61AB0.028 (LAMA2002-61-2-BB/LAMA2002-53-5-BB)-B*5-1-B6-1-B16 B 59A 0.007 SynAMP43 BC 50AB0.028 Va35 BC 50A 0.022 (LAMA2002-23-3-B/LAMA2002-58-4-B)-B-B-2-3-B-B-B-1-B23 BC 50A 0.013 (LAMA2002-22-1-B-B-B-B/LAMA2002-1-5-B-B-B-B)-2-1-B-1-1-1-B19 BC 49A 0.005 (LAMA2002-35-2-B-B-B-B/CG44)-1-3-B-1-1-B24 CD 46A 0.010 GT1216 CD 45AB0.030 Tx772-B-B-B-B-B-1-B14 CD 43A 0.012 GT1214 CD 43A 0.019 (CML450-B/Tx110)-B-3-B-1-B-B-1-1-B18 CDE 43A 0.013 (CML288/NC300)-B-9-B1-B-B-B-B-B-B-B-B14-B13 CDE 41A 0.015 (((B104/NC300)x(CML 415/B104))-4-2-B-B-B/LAMA2002-22-3-B-B1)-B-B- B-B-B CDEF 41A 0.014 Mp718 CDEFG 40A 0.014 Mp10:127 DEFGH 36A 0.018 ((Tx740) ; LAMA2002-12-1-B-B-B)-B10-B9 DEFGHI 35A 0.020 GTA2R-1B-1B-1B DEFGHI 35A 0.013 GT603 DEFGHIJ 35A 0.019 Mp04:110 EFGHIJK 31A 0.007 FAW 1430 FGHIJKL 29A 0.009 SC212m FGHIJKL 29 B0.052 Grace E-5 GHIJKLM 28 B0.052 GA209 GHIJKLM 28A 0.007 Mp10:135 HIJKLMN 27A 0.014 FAW 1382 HIJKLMN 27A 0.021 (LAMA2002-35-2-B-B-B-B/CG44)-1-3-B-B14 HIJKLMN 26A 0.025 Mp717 HIJKLMNO 24A 0.009 TX772W-B4-B13 IJKLMNO 23A 0.008 Mp719 JKLMNOP 22A 0.014 Mp04:115 KLMNOPQ20A 0.010 GT1202 LMNOPQ17A 0.022 Mp420 MNOPQ16A 0.013 Mp313E MNOPQ15A 0.016 CY-2 NOPQ15A 0.011 MP313E OPQ12A 0.011 CY-4 PQ9A 0.022 CY-1 Q8AB0.025 Mp494 Q8A 0.007 2013 inbred aflatoxin and yield trial showed good separation for yield; released lines in yellow

25.  Intermated four way crosses of Tx772, MP313E, Tx740, Mp715 (four of the most resistant public inbred lines) twice with a population size of two hundred ears and are being grown in the SERAT trial this year as well as selected for earlier maturity.  Additionally 14 eight way crosses of the most resistant lines from Marilyn's association mapping study have been developed and are being tested for yield and aflatoxin in Weslaco and College Station this summer before we select one or two for additional analysis. Synthetic Populations from Dr. Seth Murray

26. Enhancing resistance to aflatoxin contamination by molecular marker assisted selection USDA/ARS Corn Host Plant Resistance Research Unit Mississippi State University

27. Investigating the function and expression of genes associated with resistance to aflatoxin contamination

28.  Identified promising new sources of resistance from GEM germplasm.  Association mapping identified genes for aflatoxin resistance. Dr. Paul Williams, Dr. Marilyn Warburton and Dr. Matt Krakowsky, USDA-ARS

29.  Project: Reducing pre-harvest losses from aflatoxin in maize production through integrated breeding and pest management strategies  PD: Seth Murray  Co-PDs: Tom Isakeit, Wenwei Xu, Michael Brewer, Marilyn Warburton, Paul Williams,  Collaborators: Gary Odvody, David Gibson, Matthew Krakowsky, Xinzhi Ni, and Brian Scully.  Without AMCOE funding, we would not have received this USDA-AFRI funding. THANK YOU! $1 million USDA-AFRI Grant!

30. Acknowledgement Funding from the National Corn Grower Association/AMCOE, Texas Corn Producers Board, USDA and other agencies are greatly appreciated.

31. Thank You