Breeding Drought Tolerant Corn and Elucidating the Mechanisms of the Corn-Aspergillus flavus Interactions as Influenced by the Environment Baozhu Guo and Bob Kemerait University of Georgia, Department of Plant Pathology USDA-ARS, Crop Protection and Management Research Unit Tifton, GA AMCOE 2013 research project reports, February 25, 2014, Grand Hyatt, San Antonio, TX

Acknowledgement USDA-ARS Brian Scully Xinzhi Ni Joe Knoll Ted Webster Matt Krakowsky, (Raleigh, NC) Paul Williams (Mississippi State) Marilyn Warburton (Mississippi State) Hamas Abbas (Stoneville, MS) University and Industry Dewey Lee Anton Coy Wenwei Xu (TX) Daniel Gorman (Pioneer) Seth Murray (TX) Zhi-yuan Chen (LSU) Ashli Brown (MS) Nick Smith

Aflatoxin Resistance/ $Yield$ Drought tolerance/ Three factors have been primarily responsible: 1) drought/yield/quality/aflatoxin; 2) aflatoxin contamination/quality/safety/drought; 3) insect pressure/yield/aflatoxin. Genes/markers What Do You Want Us to Do? Genes/markers

Plant Breeding: Host Plant Resistance Reduced aflatoxin through increased resistance to Aspergillus flavus and subsequent aflatoxin contamination Reduced aflatoxin through increased tolerance to Drought Stress Understand the role of oxidative stress caused by drought and high temperature in aflatoxin production and its remediation by the host-corn and the pathogen-A. flavus. Yield Integrated Strategies to Manage Aflatoxin Contamiantion Improve agronomic performance (including earliness as an avoidance mechanism) Planting date Cropping system and bio-control We need all these tactics combined together Objectives and Goals:

Diverse Germplasm Used in Breeding Programs Source General QualityOther General Traits. Latin-American:+Tropical, Late, photoperiod, Insect Resistance (BZ) CIMMYT-Mexico+++Tropical, Agronomics, Drought, Insect Resistance South African+Drought, White flints, Subtropical Mediterranean +++Drought, Agronomics, Yield China++Agronomics, Yield & Aflatoxin (TUN & CY) GEM Materials+++Agronomics, Yield & Aflatoxin X-PVPs+++Agronomics, Yield Holden’s Foundation+++Agronomics, Yield Hawaiian+/++Disease Resistance & Aflatoxin ARS-NC+++Agronomics, Yield ARS-MS/LA+++Yield, Insect Resistance & Aflatoxin ARS-Tifton++Insect Resistance & Aflatoxin (CEW,FAW & GT- MAS)

InbredYear OriginPedigree GT TiftonGT-MAS:gk GT TiftonGT-MAS:gk GT-603* 2011 Tifton GT-MAS:gk GT Tifton DK 888:N11 (GEM) * Indicates “best” in group compared to the Research Standard: ‘MP 313E ‘ Plus 3 Inbreds from Texas A & M: Tx736, Tx739, Tx740 6 Inbreds from ARS New Orleans & IITA: TZAR 101 –TZAR Inbreds from ARS Mississippi State: MP718 & MP 719 Recent Inbred Releases

GT Series GT Series – Tifton, GA HBA-1 X GT-603 GT-603 GT-601, GT-602 and GT-603 all derived from same population GT-MAS:gk GT-603 appears to be the “better” of the three, but all have similar levels of resistance. GT-603 was comparable to MP 313E for aflatoxin resistance but not superior. All three Yellow dent, but GT-603 has dark cob and deeper yellow kernels and tassels 10 days earlier than MP 313E. GT-603 appeared to show better heterosis when test crossed to Stiff Stalk inbred B73 rather than Non Stiff Stalk inbred Mo17, suggesting it fits in NSS group. HybridInbred

DK888:N B B*3 Raleigh 2014 release: “GT 888”- Tifton/ Raleigh DK888 Hybrids Derived from GEM Program, Yellow Dent, Originally from Thailand Improved Aflatoxin resistance, but not superior to MP 313E (96 ppb) Tassel time earlier than Mp313E and nearly equivalent to GT-603. Fits better in Non Stiff Stalk (NSS) Group ( LH51, Mo17, Oh43). Heterotic with “Stiff Stalk Group” (SS) (B14, B73, LH132). Hybrids Commercial Hybrids Inbred

Near-future release: “GT A2R” and SynAMP43- Tifton Derived from same population as GT-601, GT-602 and GT-603 Improved aflatoxin resistance, but not as good as GT-603 Has broader General Combining Ability and produces hybrids with higher yields than others from GT Population Fits better in “Stiff Stalk Group” (SS) (B14, B73, LH132); Heterotic with Non Stiff Stalk (NSS) ( LH51, Mo17, Oh43). Hybrids Commercial Hybrids

DesignationPedigreeRepTiftonMississippi Commercial CK 1P31P Commercial CK 2P31G Commercial CK 3DK Commercial CK 4BH8740VTTP Commercial CK 5BH8910RR/HX Commercial CK 6BH9051RR GT-1Hi33 x Ni GT-2HBA x GT GT-3CY1 x GT GT-4LH132 x SynAMP GT-5LH132 x GTA2R GT-6Hi27 x GT GT-7LH51 x SynAMP Average SERAT (Southeast Region Aflatoxin Test) TEST-Afl (ppb)

2013 Hybrid Performance Test, 8 reps x 5 ears, Tifton Hybrid Ear Length (cm) Rows Kernels Ear Worm Damage (cm) Other Damage 1 Total Wt. (g) 2 Kennel Wt. (g) Yield (bu) Total Aflatoxin (ppb) LH 132 x GT LH 132 x Syn Am1 (P43) Hi 41 x Mo GT A2 R x B LH 51 x GT CY 1 x A LH132 x GT A2 R Lo 964 x A632 (green) GP282 x GT GT603 x A Syn AM1 (P43) x Mo GT603 x A Lo 964 x A632 (red silk) CY 2 x A Syn AM1 (P43) x A Lo 1016 x A Grace E-5 x GT Hi 27 x Mo LH 51 x Syn AM1 (P43) DKC Lo 1016 x A GT A2 R x Mo GT A 2R x A Tun 18-2 x GT LH 51 x GT CY 1 x A Lo 964 x A P2023HR GP 280 x GT CY 2 xMo

Hybrid Ear Length (cm) Rows Kernels Plant Stand Ear Worm Damage Other Damage Total Wt. (g) Kennel Wt. (g) Yield (Bu) GT A2 R x CY LH 132 x Mo CY2 x B GP 280 x Syn AM1 (P43) Lo 964 x GP GP 280 x GT 603 (P50) GT 603 (P50) x DK 888N GT 603 (P50) x DK 888N Syn AM 1 (P43) x B Lo 1016 x CY DKC GT 601 x AT Lo 1016 x CY LH 51 x Syn AM1 (P43) LH 132 x GT A2 R Lo 964 x CY GP 280 x Syn AM1 (P43) GT 603 (P50) x GP CY1 x GT 603 (P50) Syn AM 1 (P43) x LH Lo 1016 x CY LH 51 x B CY1 x B Lo 964 x A632 RED SILK Syn AM 1 (P43) x Mo LH 132 x Syn AM1 (P43) GP 282 x Syn AM1 (P43) P2023 HR Syn AM 1 (P43) x LH LH 51 x GT A2 R Hi 41 x Mo GT 603 x LH LH 51 x GT Lo 964 x Syn AM1 (P43) GT A2 R x Mo GP 282 x Mo CY1 x GT A2 R GT 603 (P50) x CY Hybrid Performance Test, 4 reps x 5 ears, Tifton

iTRAQ-based proteomics of corn lines tolerant and sensitive to drought stress isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology

Figure 1. Experimental design and iTRAQ-based workflow showing different steps describing corn kernels response to drought stress treatments. Maize drought tolerant line Lo964 Maize drought sensitive line B73 ControlDroughtControlDrought Protein extraction (Trichloroacetic acid/Acetone) Solubilization (6M urea, 50mM TEAB, 2% CHAPS) Reduction with DTT and Alkylation with IAA Trypsin digestion Peptide iTRAQ Labeling Peptide mixture SCX seperation and fractionation nLC-MS/MS and Data analysis Quantification: Bradford method SDS-PAGE Two biological replicates Two biological replicates and four technical replicates Analysis of differential proteome patterns and functional annotation Drought treatment for 7 days at 20 DAP

Lo964B73 Protein-protein interaction analysis: Transcription and protein folding Carbohydratemetabolism Proteindegradation Redoxhomeostasis ABA and Lea protein Signal transduction and protein metabolism Carbohydratemetabolism

DNARNA Protein Modified protein Transcription AP2-EREBP-type transcription factor; fibrillarin-2 Protein synthesis ribosomal protein; QM-like protein; histone deacetylase 6 Protein folding, assembly HSPs; TCP-1; histone deacetylase 6 Protein degradation proteasome subunit alpha type; proteasome subunit alpha type 1 Figure 7. transcription and protein metabolism B73 Lo964 B73 Degradation

why does Aspergillus flavus produce aflatoxins”? Hypothesis: given the role of oxidative stress (drought) in the promotion of aflatoxin biosynthesis, the hypothesis has been proposed that aflatoxin may function as a form of antioxidant protection to the fungus from the oxidative stress caused by the “Hot” and “Dry” environment. Jake Fountain, Ph.D. Student

Increasing H 2 O 2 Concentration Stimulates Aflatoxin Production 0 mM0.1 mM5 mM10 mM AFB 1 A. Flavus Strain 3557 (Toxigenic) Non-Concentrated YES Culture Media – 7 Days Hydrogen Peroxide Concentration Gradient = 0, 0.1, 5, 10, 50, 100mM 50mM and 100mM concentrations were lethal. 3% H 2 O 2 : 0mL 0.1mL/1000mL 5.7mL/1000mL 11.3mL/1000mL

Fungal Biomass is Affected by High H 2 O 2 Concentration and Aflatoxin Production n = 6 Error Bars ± SD ** p < ** A. flavus strains A1 (AF -) and A9 (AF +) were cultured on PDB with H 2 O 2 concentration gradient from 0 to 10mM for 3 Days. Both strains at 25mM incubated for 6 days due to slow growth.

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