Aew2005 PDF

Proceedings of the 2005 Annual
Multicrop Aflatoxin/Fumonisin
Elimination & Fungal Genomics
Workshop
October 24 26, 2005
Raleigh, North Carolina USA
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Cotton Incorporated
Arizona Cotton Research
and Protection Council
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C
R
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The Cotton Foundation
National Cottonseed
Products Association
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Table of Contents
Introduction: . . . . . . . . . . . . . . . . . . . . . . . . . 13
Agenda . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5TH ANNUAL FUNGAL GENOMICS WORKSHOP
Moderator: Roy Cantrell, Cotton Incorporated
PLATFORM PRESENTATIONS
Finding Target Genes for Better Control of Aspergillus
Jong H. Kim, Bruce C. Campbell, Jiujiang Yu, Gregory S. May, Kathleen L. Chan, Gary A.
Payne, Deepak Bhatnagar and Thomas E. Cleveland
. . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Comparative Genomic Analysis of Secondary Metabolite Gene Clusters of Closely
Related Aspergilli
William C. Nierman, Natalie D. Fedorova, Catherine M. Ronning, Jennifer Wortman,
Masayuki Mashida, Jiujiang Yu, Thomas E. Cleveland, Deepak Bhatnagar and Gary Payne
. . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Aspergillus flavus Genomics in Discovering Genes Involved in Aflatoxin Biosynthesis
J. Yu, J.R. Wilkinson, W.C. Nierman, H.S. Kim, G.A. Payne, B.C. Campbell, D. Bhatnagar,
and T. E. Cleveland
. . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Mining Expressed Sequence Tags (ESTs) Leads to Identification of Putative FUM Cluster
Transcription Factor
Daren W. Brown, Robert A.E. Butchko, Mark Busman and Robert H. Proctor
. . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Release of the Aspergillus flavus Genome Sequence
Gary A. Payne, B. Pritchard, Jiujiang Yu, William C. Nierman, Ralph Dean, Deepak
Bhatnagar and Thomas E. Cleveland
. . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Production of Cyclopiazonic Acid, Aflatrem and Aflatoxin is Regulated by veA, a Gene
Necessary for Sclerotial Formation in Aspergillus flavus
R.M. Duran, J.W. Cary, and A.M. Calvo
. . . . . . . . . . . . . . . . . . . . . . . . . . . 40
PANEL DISCUSSION: Fungal Genomics Workshop
Panel Chair: Gary Payne, North Carolina State University . . . . . 41
POSTER PRESENTATIONS
Evolutionary Processes in the Aflatoxin Gene Cluster in Aspergillus
I. Carbone, J.L. Jakobek, E.H. Moussa,, J.E. Cox and B.W. Horn
. . . . . . . . . . . . . . . . . . . . . . . . . . . 43
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Differential Gene Expression Levels for Aspergillus flavus Resistance in Two Inbred
Maize Lines
R. Y. Kelley, D. L. Boykin, L. K. Hawkins and W. P. Williams
. . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Enhanced Activity of Fungicides by Positive Interaction with Natural Phenolic Agents:
Target-gene Based Bioassays for Control of Aspergilli
Jong H. Kim, Bruce C. Campbell, Jiujiang Yu, Noreen Mahoney, Kathleen L. Chan, Russell
J. Molyneux, Deepak Bhatnagar, Thomas E. Cleveland, Gregory S. May and Gary A. Payne
. . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Deletion of GBP1, a Gene Encoding a Monomeric G Protein, De-represses Fumonisin
Biosynthesis in Fusarium verticillioides
U.S. Sagaram and W.B. Shim
. . . . . . . . . . . . . . . . . . . . . . . . . . . 46
A Link between Rho-Signaling and Aflatoxin Biosynthesis in Aspergillus flavus
D. Ryan Georgianna, Michael S. Price and Gary A. Payne
. . . . . . . . . . . . . . . . . . . . . . . . . . . 47
The NADH oxidase, NadA, and its Role in Aflatoxin Biosynthesis
Carrie Jacobus, Gary Payne and Niki Robertson
. . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Metabolic Profiling of Aspergillus flavus during Aflatoxin Biosynthesis
Norm Glassbrook and Gary A. Payne
. . . . . . . . . . . . . . . . . . . . . . . . . . . 49
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ANNUAL FUMONISIN ELIMINATION WORKSHOP
Moderator: Larry Antilla, Arizona Cotton Research and Protection Council
Kernel Constituents Induce Fumonisin Production during Colonization by Fusarium
verticillioides
Charles Woloshuk and Burt Bluhm
. . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Genetics and Breeding of Host Resistance to Fusarium Ear Rot and Fumonisin
Contamination
J.B. Holland
. . . . . . . . . . . . . . . . . . . . . . . . . . . 52
NIR Spectroscopy as a Tool for Optimizing Sorting of White Corn Kernels Contaminated
with Fumonisin
T.C. Pearson and D.T. Wicklow
. . . . . . . . . . . . . . . . . . . . . . . . . . . 53
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Maize LOX3 Gene is Required for Fumonisin Biosynthesis and Conidiation of Fusarium
verticillioides
Xiquan Gao, Won-Bo Shim, Ivo Feussner and Mike Kolomiets
. . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Toxicity Responses of Corn to the Mycotoxin Fumonisin B1 in the Absence of Fusarium
verticillioides Infection
A.M. Zimeri, L.D. Williams, R.T. Riley and A.E. Glenn
. . . . . . . . . . . . . . . . . . . . . . . . . . . 55
PANEL DISCUSSION: Fumonisin Elimination
Panel Chair: Charles Woloshuk . . . . . . . . . . . . . . . 56
QTL Mapping for Fusarium Ear Rot and Fumonisin Contamination Resistance in Two
Populations of Maize (Zea mays)
Leilani A. Robertson, Michael P. Jines, Peter Balint-Kurti, Gary A. Payne, Donald G. White,
and James B. Holland
. . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Polyketide Synthases in Fusarium verticillioides: Potential Targets to Control Fumonisin
Contamination in Corn
Robert H. Proctor, Robert A.E. Butchko, Ronald D. Plattner, Mark Busman, Daren W.
Brown, and Anne E. Desjardins
. . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Computational Studies on the Influence of Solvent on the Conformational Preferences
and Selective Recognition of Fumonisins
M. Appell, C.M. Maragos and D.F. Kendra
. . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Using Genomics Approaches to Characterize Potential Fumonisin Regulatory Genes
Robert A.E. Butchko, Robert H. Proctor, Daren W. Brown, Charles P. Woloshuk, Burton H.
Bluhm and Mark Busman
. . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Fumonisins in Maize in Guatemala, Preliminary Exposure Estimate, and Policies and
Recommendations to Minimize Exposure
Ronald T. Riley, Olga A. Torres, Edwin Palencia, L. Lopez de Pratdesaba, Anthony. E.
Glenn, Kerry ODonnell and Mario Fuentes
. . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Fusaric Acid, a Fusarium verticillioides Miasma to Bacillus mojavensis, a Biological
Control Bacterial Endophyte
Charles W. Bacon and D. M. Hinton
. . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Developmental Toxicity of Fusarium verticillioides and Fumonisin B1 in LM/Bc and CD1
Mice: Comparing the in vivo Models
Kenneth A. Voss, Ronald T. Riley, Tantiana D. Burns and Janee B. Gelineau-van Waes
. . . . . . . . . . . . . . . . . . . . . . . . . . . 63
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ANNUAL AFLATOXIN ELIMINATION WORKSHOP
SESSION 1: Crop Resistance Conventional Breeding
Moderator: Don Jones, Cotton Incorporated
Creation of Commercial Hybrids with Low Aflatoxin in Grain using Markers
Don White and Torbert Rocheford
. . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Breeding Corn Germplasm for Agronomic Performance and Reduced Aflatoxin
Contamination
Javier Betrn, Tom Isakeit, Gary Odvody and Kerry Mayfield
. . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Interaction Between A. flavus Strains and Host Plant Genotypes Across Environments
and Years
Kerry Mayfield, Tom Isakeit, Gary Odvody and Javier Betrn
. . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Application of HACCP to Control Mycotoxins in Maize Breeding Programs
David F. Kendra
. . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Characterizing Components of Insect-Based Resistance to Preharvest Aflatoxin
Contamination in Almond
T.M. Gradziel and A.M. Dandekar
. . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Genetic and Genomic Approaches to Improve Host Resistance to Preharvest Aflatoxin
Contamination in Corn and Peanut
B.Z. Guo, M. Luo, H. Chen, P. Dang, A.E. Coy, M.D. Krakowsky, D. Davis, W. Xu, X. Liang,
C. Holbrook, R.D. Lee, M. Bausher, A. Culbreath, P. Ozias-Akins and Craig K. Kvien
. . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Progress Toward Identifying New Sources of Genetic Variation Associated with Reduced
Levels of Aflatoxin Accumulation in Maize
Thomas Brooks, Matthew Krakowsky, W. Paul Williams and Gary Windham
. . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Proteomic Identification of Maize Cob Proteins that Potentially Confer Resistance to
Aflatoxin
Dawn Luthe, Olga Pechanova, Bela Peethambaran, Tibor Pechan, Susan Bridges, Leigh
Hawkins, Gary Windham and W. Paul Williams
. . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Development of Field Based Techniques for Assessing Variability Among Cotton
Cultivars in Susceptibility to Aflatoxin Contamination During the Second Phase of
Contamination
M.W. Olsen, P.J. Cotty and S. Husman
. . . . . . . . . . . . . . . . . . . . . . . . . . . 73
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Corn Hybrids with Exotic Germplasm and Low-Aflatoxin
Wenwei Xu, Jinfen Zhang, Gary Odvody, and W. Paul Williams
. . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Computational Tools for Protein Identification and Gene Ontology Annotation of the
Maize Proteome
Susan M. Bridges, Julia E. Hodges, Gregory Bryce Magee, Nan Wang, Dawn S. Luthe and
W. Paul Williams
. . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Progress in Breeding Peanut for Resistance to Preharvest Aflatoxin Contamination and
Drought
C.C. Holbrook, B.Z. Guo, P. Timper, D.M. Wilson, D. Sullivan, E. Cantonwine and C. Kvien
. . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Searching for New Resistance and Control Measures of Aflatoxin in Corn
Steven Moore, Hamed Abbas and Mark Millard
. . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Development of Aflatoxin-resistant Maize Inbreds and Identification of Potential
Resistance Markers through USA-Africa Collaborative Research
Robert L. Brown, Zhi-Yuan Chen, Abebe Menkir and Thomas E. Cleveland
. . . . . . . . . . . . . . . . . . . . . . . . . . . 78
PANEL DISCUSSION: Crop Resistance Conventional Breeding
Panel Chair: Don White . . . . . . . . . . . . . . . . . . 79
Multilocation Evaluation of Aflatoxin Accumulation in Yellow Maize Hybrids
Cody McKee, Tom Isakeit, Gary Odvody, Kerry Mayfield and Javier Betrn
. . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Southern East Regional Aflatoxin Test (SERAT)
Michael Clements, Paul Williams, Steve Moore, Matthew Krakowsky, Baozhu Guo, Don
White, Wenwei Xu, Tom Isakeit, Tom Brooks, Gary Windham, Hamed Abbas, James
Perkins, Daniel Gorman, Quinton Raab, Keith Arnold, David Smith and Javier Betrn
. . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Evaluation of CIMMYT Germplasm for Response to Aflatoxin Production in the Southern
USA
Dan Jeffers, Matt Krakowsky, Paul Williams and Javier Betrn
. . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Phenotypic and Genotypic Characterization of a RIL Maize Mapping Population for
Aflatoxin and Secondary Traits
Melanie Edwards, Monica Menz, Tom Isakeit and Javier Betrn
. . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Expression of LOX Pathway Genes in Corn Embryos Associated with Aspergillus flavus
Resistance
Alberto Camas, L. Lopez, P. Williams and D.S. Luthe
. . . . . . . . . . . . . . . . . . . . . . . . . . . 84
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Breeding for Increased Resistance to Fusarium verticillioides in Maize
Magen Starr, Leilani Robertson, James Holland and Gary Payne
. . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Quantitative Expression Analysis of Adversity Resistance Genes in Corn Germplasm with
Resistance to Preharvest Aflatoxin Contamination
M. Luo, D. Davis, W. Xu, D. Lee and B.Z. Guo
. . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Peanut PR Protein, -1,3-glucanase, Induction by Aspergillus flavus and Copurification
with a Conglutin-like Protein
X. Liang, B.Z. Guo and C.C. Holbrook
. . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Corn Husk Characteristics Potentially Associated with Resistance to Aflatoxin
Contamination of Grain: A Preliminary Study
M.J. Clements and W.P. Williams
. . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Chalcone Synthase, a Gene that Influences Both Drought Response and Aflatoxin
Accumulation in Maize
M. Gerau, D. Bush, D. Davis, C. Morriss and G. Davis
. . . . . . . . . . . . . . . . . . . . . . . . . . . 89
SESSION 2: Microbial Ecology
Moderator: Phil Wakelyn, National Cotton Council
Effect of Fungal Competition on the Colonization of Wounded Peanut Seeds by
Aspergillus section Flavi from Natural Soil Populations
B.W. Horn
. . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Transfer of Aflatoxin Biocontrol Technology: Results of First Commercial Use in Peanuts
Joe W. Dorner
. . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Atoxigenic Strain Technology for Aflatoxin Control in Cotton
Larry Antilla and Peter J. Cotty
. . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Managing Aflatoxins in Cotton-Corn Rotations
Peter J. Cotty
. . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Aflatoxin Control in Pistachios: Biocontrol Using Atoxigenic Strains
Mark Doster, Themis Michailides, Peter Cotty, Dave Morgan, Lorene Boeckler, Dan Felts
and Heraclio Reyes
. . . . . . . . . . . . . . . . . . . . . . . . . . . 95
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Aflatoxin Control in Figs: Biocontrol and New Resistant Cultivars
Mark Doster, Themis Michailides, Peter Cotty, Louise Ferguson, James Doyle, David
Morgan, Lorene Boeckler, Dan Felts and Heraclio Reyes
. . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Identification of Bacterial Antagonists of Aspergillus flavus from California Almond
Orchards
Jeffrey D. Palumbo, James L. Baker and Noreen E. Mahoney
. . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Biological Control of Aspergillus flavus by a Saprophytic Yeast Strain in Tree-Nut
Orchards: Progress in 2005
Sui Sheng Hua
. . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Cultural Conditions Promoting Chitinase Production in Gliocladium catenulatum
David F. Kendra, Michael J. Muhitch, Amber Anderson and Cesaria E. McAlpin
. . . . . . . . . . . . . . . . . . . . . . . . . . . 99
PANEL DISCUSSION: Microbial Ecology
Panel Chair: Bruce Horn . . . . . . . . . . . . . . . . . 100
Influences of Crops and Geographic Features on Communities of Aflatoxin-producing
Fungi
Ramon Jaime and Peter J. Cotty
. . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Aflatoxin Contamination of Maize in Africa
Claudia Probst, Henry Njapau and Peter J. Cotty
. . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Influences of Herbicides on Release of Atoxigenic Strains
Nicholas P. Garber and Peter J. Cotty
. . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Screening of Atoxigenic Aspergillus flavus Isolates for Ability to Inhibit Aflatoxin B1
Production by Toxigenic Aspergillus flavus
A. Jha, R. Sweany and K.E. Damann
. . . . . . . . . . . . . . . . . . . . . . . . . . . 105
SESSION 3: Crop Resistance Genetic Engineering
Moderator: Keerti Rathore, Texas A&M University
Gene-based Antifungal Strategies in Peanut
Ye (Juliet) Chu, Paola Faustinelli, Laura Ramos, Kanniah Rajasekaran, Jeff Cary, Corley
Holbrook and Peggy Ozias-Akins
. . . . . . . . . . . . . . . . . . . . . . . . . . . 107
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Transgenic Peanuts with Enhanced Resistance to Aspergillus flavus
Arthur K. Weissinger
. . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Identification, Characterization and Antifungal Activities of Silk Proteins in Aspergillus
flavus Resistant and Susceptible Corn Inbreds
Bela Peethambaran, Gary L. Windham, Leigh Hawkins, Paul Williams and Dawn S. Luthe
. . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Silencing the Expression of RAP Genes in Maize and the Effect on Host Resistance
against Aspergillus flavus Infection and Aflatoxin Production
Zhi-Yuan Chen, Robert L. Brown, Thomas E. Cleveland and Kenneth E Damann
. . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Genetic Engineering of Cotton for Resistance to Phytopathogens including Aspergillus
flavus
Kanniah Rajasekaran, Mauricio Ulloa, Bob Hutmacher, Jeff Cary, Jesse M. Jaynes and
Thomas Cleveland
. . . . . . . . . . . . . . . . . . . . . . . . . . . 111
PANEL DISCUSSION: Crop Resistance Genetic Engineering
Panel Chair: Arthur Weissinger . . . . . . . . . . . . . . . 112
SESSION 4: Crop Management and Handling, Insect Control and Fungal Relationships
Moderator: Pat OLeary, Cotton Incorporated
Update on Validation and Distribution of a Computer Program for Predicting Mycotoxins
in Midwest Corn
Patrick F. Dowd
. . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Mechanisms of Preharvest Aflatoxin Contamination in Peanut Infected by Root-Knot
Nematodes
Patricia Timper, Corley Holbrook and Dave Wilson
. . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Experimental Use of the Pear Ester Kairomone to Improve Codling Moth Control in
Walnuts
D.M. Light, K.M. Reynolds, P. Bouyssounouse and B.C. Campbell
. . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Liberty Link and Urea on Aflatoxin and Fumonisin Levels in Corn
H. Arnold Bruns and H. K. Abbas
. . . . . . . . . . . . . . . . . . . . . . . . . . . 117
PANEL DISCUSSION: Crop Management and Handling, Insect Control and Fungal
Relationships
Panel Chair: Pat Dowd . . . . . . . . . . . . . . . . . . 118
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Anthocyanins from Petunia Floral Structures that Inhibit Corn Earworm Development
Eric T. Johnson, Patrick F. Dowd and Mark A. Berhow
. . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Ground-Based Remote Sensing for Rapid Selection of Drought and Aflatoxin Resistant
Peanut Genotypes
D.G. Sullivan and C.C. Holbrook
. . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Correlations Between Biotic Stresses and Aflatoxin Contamination in Maize
Matthew Krakowsky, Xinzhi Ni and Richard Davis
. . . . . . . . . . . . . . . . . . . . . . . . . . . 121
SESSION 5: Detection, Extraction, and Analysis of Aflatoxins; Potential Use of Natural
Products for Prevention of Fungal Invasion and/or Aflatoxin Biosynthesis in Crops
Moderator: Tom Wedegaertner, Cotton Incorporated
Distribution of Aflatoxin in Non-irrigated Peanuts
Thomas F. Schatzki and Martin S. Ong
. . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Inhibition of Aspergillus flavus Aflatoxin Biosynthesis by Antioxidant Phytochemicals
Occurring in Tree Nuts
Russell J. Molyneux, Noreen Mahoney, Bruce C. Campbell and Jong H. Kim
. . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Biochemical and Genetic Analysis of Gallic Acid in Walnuts in Relation to Aflatoxin
Accumulation
Ryann M. Muir, Elizabeth Ingham, Sandra Uratsu, Gale McGranahan, Charles Leslie,
Noreen Mahoney and Abhaya Dandekar
. . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Inhibition of Aflatoxin Production by Compounds in Corn Seeds
G.A. Payne, R.A. Holmes and R.S. Boston
. . . . . . . . . . . . . . . . . . . . . . . . . . . 126
PANEL DISCUSSION: Detection, Extraction and Analysis of Aflatoxins; Potential Use of
Natural Products for Prevention of Fungal Invasion and/or Aflatoxin Biosynthesis in
Crops
Panel Chair: Russell Molyneux . . . . . . . . . . . . . . . 127
Identification of Two Maize Seed Compounds that Influence Aflatoxin Biosynthesis
Robert A. Holmes, Norman J. Glassbrook, Rebecca S. Boston and Gary A. Payne
. . . . . . . . . . . . . . . . . . . . . . . . . . . 128
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A New Peanut Phytoalexin with Stilbene and Tetronic Acid Moieties
V.S. Sobolev, S.T. Deyrup and J.B. Gloer
. . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Examination of Error Components Associated with Quantification of Aflatoxin in Ground
Corn Grain with In-house CD-ELISA
M.J. Clements, G.L. Windham, C.M. Maragos, W.P. Williams, T.D. Brooks, L.K. Hawkins
and H.M. Gardner
. . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Using Hyperspectral Technology to Measure Fungal Growth and Assess Mycotoxin
Contamination of Corn
Z. Hruska, H. Yao, K. DiCrispino, K. Brabham, D. Lewis, J. Beach, R.L. Brown and T.E.
Cleveland
. . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Participants . . . . . . . . . . . . . . . . . . . . . . . . 132
Author Index . . . . . . . . . . . . . . . . . . . . . . . . 141
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Introduction:
Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop 2005
Raleigh, NC
!l any ol us wcrc bccoming smug in bclicving that aatoxin would ncvcr bc a rcal problcm hcrc in thc US,
thc cvcnts starting at Christmas 200 should havc cmphatically changcd our minds. Vc may givc thanks
and rclax somcwhat that thcrc was no human involvcmcnt with aatoxin poisoning, but consumcrs did
bring aatoxin into thcir homcs in contaminatcd lood lor thcir dogs. Tc latcst statcmcnts lrom Corncll
Univcrsity Collcgc ol \ctcrinary Mcdicinc whcrc thc dcnnitivc diagnoscs wcrc madc arc that thcy bclicvc
that ovcr !00 dogs havc dicd in rcccnt wccks. Tis lollows cascs ol human ingcstion ol toxic amounts ol
aatoxin and rcsulting morbidity and mortality lrom cating homc grown corn in Kcnya in 2004.
Tc prcscntations at thc 200 Aatoxin Vorkshop which locuscd on prcharvcst aatoxin control
should givc hopc that aatoxin can bc climinatcd as a scrious problcm during production ol susccptiblc
crops in thc U.S. !nnatc crop rcsistancc and good crop production practiccs nccd to bc, at lcast, cqual
partncrs with statistically valid sampling, rcgular, scnsitivc and accuratc product assays, propcr commodity
handling and proccssing practiccs and/or thc usc ol absorbing clays in product lormulations in assuring
lood product salcty.
Highlights of the Presentations of the Workshop follow:
Fumonisins
Fusarium verticillioides is a lungal pathogcn ol maizc throughout thc world. Tc lungus can inlcct all stagcs
ol maizc dcvclopmcnt and almost cvcry tissuc typc ol thc maizc plant, thus causing sccdling blight, root
rot, stcm rot, and kcrncl rot. ach ol thcsc discasc manilcstations can rcsult in scvcrc cconomic losscs. Tc
lungus also can cocxist rclativcly pcacclully with maizc as an cndophytc, maintain a biotrophic growth
habit throughout thc cntirc growing scason ol thc plant, and causc asymptomatic inlcctions ol kcrncls.
uring thc colonization ol maizc kcrncls, F. verticillioides produccs toxic sccondary mctabolitcs known as
lumonisins. Fumonisins causc a rangc ol spccicsspccinc hcalth cccts whcn ingcstcd and arc suspcctcd
to causc canccr and birth dclccts in humans. At thc workshop scssion on Fumonisin limination, nvc
prcscntations locuscd on thrcc important rcscarch arcas: brccding lor rcsistancc, undcrstanding thc basis
lor lumonisin production, and idcntilying lumonisincontaminatcd grain along thc markcting supply
chain.
For many ycars, pathologists havc known that thc amount ol lumonisin contamination in maizc docs
not corrclatc to thc scvcrity ol kcrncl damagc causcd by thc pathogcn. Maizc brccdcrs who havc studicd
thc inhcritancc ol rcsistancc havc contcndcd that discasc scvcrity, which is casy to scorc, and lumonisin
contamination, which is cxpcnsivc to analyzc, arc scparablc traits. Holland prcscntcd rcsults lrom a study
ol two maizc brccding populations, in which hc cxamincd discasc scvcrity (rot) and lumonisin contcnt.
8ascd on his rcsults, Holland madc thc casc that thc most cconomical mcthod to brccd lor rcsistancc to
lumonisin contamination would bc to makc sclcctions bascd on discasc scvcrity.
nc approach toward discovcring potcntial targcts lor rcsistancc is to study thc basis ol susccptibility.
Trcc prcscntations in thc scssion wcrc in this catcgory. First, Zimcri prcscntcd cvidcncc that sphingolipid
mctabolism in maizc roots is acctcd by lumonisin, suggcsting that toxin production has a rolc in thc
pathogcncsis ol maizc sccdlings. Shc prcscntcd cvidcncc that trcatmcnt ol sccdlings with lumonisin
causcd symptoms similar to pathogcninlcctcd sccdlings. !ntcrcstingly, thc anccstors to maizc, tcosintc
and Tripsacum, wcrc morc scnsitivc to lumonisin. Sccondly, Kolomicts prcscntcd cvidcncc that suggcsts
thc LOX gcnc in maizc, which cncodcs a 9lipooxygcnasc, is associatcd with lumonisin production
in discascd kcrncls. Kcrncls lrom a maizc linc with a mutatcd lox supportcd growth ol thc pathogcn
but lumonisin production and conidiation wcrc scvcrcly rcduccd. Tc rcsult suggcsts that latty acid
hydropcroxidcs, which arc thc products ol 9lipooxygcnasc, havc a rolc in rcgulating both dcvclopmcnt
and sccondary mctabolism in thc pathogcn. Tirdly, Voloshuk prcscntcd cvidcncc that lumonisin
production in F. verticillioides is associatcd with thc mctabolism ol cndospcrm starch. Kcrncls with rcduccd
amylopcctin duc to immaturity or mutations in starch synthcsis do not support lumonisin production by
thc pathogcn. Kcy lor thc lungus is thc cxprcssion ol alphaamylasc, which dcgradcs amylopcctin to yicld
lumonisininducing alpha!,6glucosidcs.
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To assurc that thc quality ol grain is maintaincd along thc supply chain lrom thc produccr to thc
cnduscr, ncw mcthodology lor tcsting grains is nccdcd, and tcchnologics dcvclopcd in ncarinlrarcd
and rccctancc (N!R) spcctroscopy havc bccomc important tools. Vicklow dcscribcd thc application
ol N!R tcchnology to idcntily and sort molddamagcd and mycotoxincontaminatcd grain. His rcsults
showcd that a N!R sorting dcvicc that uscs two spccinc wavclcngths to asscss kcrncls was vcry ccctivc in
rcmoving highly damagcd and lumonisincontaminatcd kcrncls.
Conventional Breeding: Resistance to Aflatoxin Production
Conccrtcd coopcrativc cort and optimization ol cvaluation tcchniqucs arc rcsponsiblc lor thc signincant
progrcss now cvidcnt in thc dcvclopmcnt ol commcrcially acccptablc varictics with low lcvcls ol aatoxin
in thc commodity at harvcst. Ncwly crcatcd rcsistant varictics ol corn, pcanut and almond arc at thc carly
stagcs ol commcrcialization. !dcntincation ol plant gcnotypcs with low lcvcls ol aatoxin in sccds at
harvcst is rclativcly casy comparcd with thc problcm ol moving rcsistancc into a commcrcially acccptablc
varicty that has thc various quality traits and yicld that arc dcmandcd by produccrs. Gcncrally, thc corn,
pcanut and almond varictics dcvclopcd in this rcscarch also havc thc dcsircd commcrcial traits and will
likcly bc commcrcially uscd in thc ncar luturc.
Scvcral projccts arc continuing to idcntily ncw sourccs ol rcsistancc lor corn whcrc thcrc is a widc
varicty ol publicly availablc gcrmplasm availablc lor cvaluation that may yicld uniquc sourccs ol rcsistancc.
Additional sourccs ol rcsistancc arc ol valuc cspccially whcn thcy havc high lcvcls ol rcsistancc controllcd
by rclativcly lcw gcncs. Pcrhaps thc grcatcst challcngc ol convcntional brccding is cvaluating lor rcsistancc
and/or susccptibility in thc ncld.
Rcsistancc in corn, pcanuts, almonds and cotton can bc succcsslully idcntincd whcn cnvironmcntal
conditions arc conducivc lor aatoxin production. Vith pcanuts, modincation ol thc cnvironmcnt can
crcatc morc lavorablc conditions lor aatoxin production. Corn scicntists havc joincd togcthcr to cvaluatc
hybrids crcatcd in thc various rcscarch projccts ovcr a numbcr ol locations utilizing dicrcnt inoculation
tcchniqucs. Tis coopcrativc cort has bccn cxtrcmcly valuablc in charactcrizing thc lcvcl ol rcsistancc as
wcll as thc agronomic charactcristics ol ncwly dcvclopcd rcsistant hybrids. Convcntional brccding can also
idcntily crop charactcristics than arc strongly associatcd with low aatoxin accumulation and sclcct lor
thosc charactcristics. !n many cascs, thc associatcd traits arc casicr to sclcct lor and havc highcr hcritability
that aatoxin accumulation pcr sc. Pcrhaps thc grcatcst succcss has bccn with almond whcrc aatoxin
contamination is strongly associatcd with inscct damagcd kcrncls. Trcc dicrcnt major componcnts
ol inscct rcsistancc havc bccn succcsslully uscd in brccding to rcducc aatoxin. Advanccd sclcctions ol
almond arc dcmonstrating vcry low lcvcls ol inscct damagc and thcrclorc low aatoxin (Gradzicl).
Vith pcanut low lcvcls ol aatoxin has bccn associatcd with drought tolcrancc and rcsistancc to thc
pcanut root knot ncmatodc. Pcanut varictics havc bccn dcvclopcd with drought rcsistancc and havc
dcmonstratcd lowcr aatoxin contamination in multiplc cnvironmcnts. !t also is ncccssary to havc
rcsistancc to tomato spottcd wilt virus in a commcrcial pcanut varicty. Rcccntly advanccd brccding lincs
with rcsistancc to both virus and ncmatodc as wcll as acccptablc yicld and gradc havc bccn dcvclopcd
(Holbrook). Rcsistancc to aatoxin accumulation in corn has also bccn associatcd with rcsistancc to
drought strcss and corn carworm. \arictics sclcctcd lor rcsistancc to drought and carworm rcsistancc havc
lowcr aatoxin contamination in thc grain.
Convcntional brccding is now incorporating thc morc rcccntly dcvclopcd molccular biology tcchniqucs
to cnhancc chanccs ol succcss. Molccular markcrs lor corn arc bcing uscd to translcr chromosomc arcas
associatcd with gcncs conditioning rcsistancc into commcrcially acccptablc corn inbrcds. Tis is highly
advantagcous bccausc cvaluation lor aatoxin accumulation in grain is not ncccssary at cvcry cyclc ol
brccding. Molccular markcrs arc grcatly cnhancing thc succcsslul movcmcnt ol gcncs lrom a sourcc
ol rcsistancc with poor agronomic charactcristics into commcrcially uscd inbrcds. Rcsistancc has bccn
incorporatcd into commcrcially uscd inbrcds and hybrids that will bc cvaluatcd in prccommcrcial trials
ovcr a numbcr ol locations in 2006.
Gcnomic and protcomic tcchniqucs arc bcing utilizcd with corn and pcanut to comparc rcsistant and
susccptiblc gcnotypcs to attcmpt to idcntily charactcristics ol thc rcsistant gcnotypcs that arc associatcd
with low aatoxin. Ultimatcly cach potcntial mcchanism associatcd with rcsistancc will nccd to bc crosscd
into common gcnctic backgrounds and cvaluatcd in thc ncld which will takc scvcral ycars and considcrablc
cort. !l succcsslul, thcsc tcchniqucs ocr a possibility ol idcntilying uniquc molccular markcrs to bc uscd
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in convcntional brccding, and thcy may also idcntily actual gcncs lor rcsistancc. !n thc luturc it is highly
likcly that gcncs conditioning dicrcnt mcchanisms ol rcsistancc can bc pyramidcd into commcrcially
acccptablc varictics rcsulting in cxtrcmcly low lcvcls ol aatoxin.
Control ol aatoxin contamination by gcnctic rcsistancc has a major advantagc in that it is costccctivc
to thc produccr, cnvironmcntally lricndly, and acccptablc to thc gcncral public. !t is highly likcly that thc
tcchniqucs and proccdurcs uscd in thc dcvclopmcnt ol varictics with low aatoxin will bc modincd and
uscd to dcvclop varictics that control othcr mycotoxins.
Fungal Genomics
A numbcr ol inroads wcrc madc ovcr thc past ycar in improving our undcrstanding ol thc gcnctic basis
and lunctional gcnomics ol lumonisin and aatoxin production. Vc arc sccing incrcascd usc ol microarray
analyscs to gct a bcttcr picturc ol thc gcnctic proccsscs involvcd in thc inlcctivity and toxin production by
thcsc two agriculturally important lungi.
Vith rcgard to lumonisin, prcliminary analysis has lound a numbcr ol candidatc lumonisin rcgulatory
gcncs in Fusarium verticillioides (8rown). Tc gcnc GBP was idcntincd as bcing associatcd with
thc rcgulation ol lumonisin in thc F. verticillioides porobably indcpcndcnt ol thc FUM gcnc clustcr
(Sagram).
!n Aspergillus favus, it was lound that oxidativc strcss and antioxidants acct aatoxin biosynthcsis,
suggcsting thc antioxidativc rcsponsc systcms ol A. favus arc targcts lor control. Combincd trcatmcnt ol
lungi with phcnolics and inhibitors ol thc mitochondrial rcspiratory systcm ccctivcly supprcsscd growth
ol A. favus. Targcting gcncs in othcr antioxidativc rcsponsc, MAPK or vacuolar H()ATPasc (\
ATPasc) systcms should grcatly improvc mcthods lor lungal control using combinations ol compounds
(Kim). Tc gcnc veA rcgulatcs both, lormation ol rcsistant structurcs (sclctoria), and biosynthcsis ol
aatoxin, and othcr toxins, in A. favus. !n a veA mutant cxprcssion ol AfR and aatoxin was supprcsscd.
Targcting veA could dccrcasc A. favus survivability by accting sclcrotial dcvclopmcnt. Homologucs ol
veA wcrc lound across lungal gcncra indicating thc possibility ol targcting this gcnc lor broad spcctrum
lungal control (uran). A componcnt ol a signaling pathway that may modulatc aatoxin production in
A. favus was idcntincd. Tis idcntincd gcnc had thc grcatcst similarity to rdi, a ycast gcnc that cncodcs
a Rhoguanidinc nuclcotidc dissociation inhibitor (RhoG!). clction ol this gcnc causcd a scvcrc
growth dclccts ol A. favus on minimal mcdia, a modcratc dclcct on complctc mcdia, and a tcmpcraturc
scnsitivc phcnotypc. Tis gcnc may play a ccntral rolc in combining A. favus protcins AR and RasA lor
lacilitating signaling control ol aatoxin production through a Rhomcdiatcd pathway (Gcorgianna).
Microarray analyscs wcrc uscd as a mcans lor cstablishing a lramcwork lor sorting gcnc cxprcssion lor
aatoxin biosynthcsis on conducivc and nonconducivc conditions bascd on tcmpcraturc (Glassbrook).
thcr microarray cxpcrimcnts suggcstcd that thc gcnc nadA is uprcgulatcd by AfR to supply NA

colactors lor thc aatoxin biosynthcsis. Howcvcr, knocking out this gcnc did not acct production ol
aatoxin suggcsting that thcrc is compcnsation lor NAH oxidasc activity or that it is not rcquircd lor
aatoxin production ( Jacobus). Microarray anaysis ol maizc lincs \a1, susccptiblc, and Mp1!1, rcsistant,
to A. favus showcd cxprcssion pattcrns lor a numbcr ol maizc gcncs whcn cxposcd to A. favus (Kclly).
Gcncs that arc putativcly involvcd in aatoxin biosynthcsis, rcgulation and signal transduction, lungal
virulcncc or pathogcnicity, strcss rcsponsc or antioxidation, and lungal dcvclopmcnt wcrc idcntincd
lrom an A. favus ST library. Tis was uscd to construct microarrays containing ovcr ,000 uniquc gcnc
amplicons. Microarraybascd gcnc pronling has thus lar idcntincd hundrcds ol gcncs that arc potcntially
involvcd in aatoxin production. Tis rcscarch is cxpcctcd to providc inlormation lor dcvcloping ncw
stratcgics lor control ol aatoxin contamination ol agricultural commoditics (Yu).
Gcnomic analysis rcscarch is undcrway to dctcrminc il gcnc clustcrs associatcd with sccondary
mctabolism ol A. favus arc within polymorphic subtclomcric domains as lound in othcr closcly rclatcd
lungi (Nicrman). A X covcragc ol thc gcnomic scqucncc ol A. favus was complctcd and prcliminarily
annotatcd as much as possiblc. A wcb browscr has bccn sct up at NC Statc allowing 8LAST scarchcs ol
gcncs, protcins and gcnomic scqucnccs ol A. favus and othcr Aspergillus spccics including alignmcnts ol
STs, and G annotations (Paync).
Rcscarch was prcscntcd suggcsting thc potcntial lor lowlcvcl rccombination and gcnc ow bctwccn
atoxigcnic and toxigcnic strains ol A. favus. How this may acct usc ol atoxigcnic strains lor biocontrol
rcmains to bc sccn (Carbonc).
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Improvement of Aflatoxin Resistance via Genetic Engineering
All ol thc currcnt rcscarch projccts scck to cithcr rcducc aatoxin contamination by rctarding Aspergillus
inlcction, or intcrlcrc with mcchanisms involvcd in toxin synthcsis. Stratcgics includc translormation
ol crop spccics with cithcr naturally occurring or synthctic antilungal gcncs, mutation brccding, and thc
dcvclopmcnt ol rcliablc molccular markcrs to improvc thc cmcicncy ol brccding programs to producc
improvcd lungal rcsistancc.
Trcc distinct projccts attcmpt to rcducc both inlcction and aatoxin contamination in pcanut
(ziasAkins). First arc transgcnic pcanuts that cxprcss an antiapoptotic gcnc, 8clxl, thc product
ol which is cxpcctcd to rcducc Aspergillus inlcction. Sccond is a mutation brccding program involving
MSmutagcnizcd pcanut populations which arc bcing studicd by T!LL!NG (Targcting !nduccd Local
Lcsions !N Gcnomcs) to idcntily gcncs that might bc manipulatcd to altcr lungal inlcction and/or
aatoxin contamination. T!LL!NG can idcntily mutants bascd on scrccning with gcnc scqucncc rathcr
than lor phcnotypc. Tc T!LL!NG tcchniquc is bcing tcstcd with an allcrgcn gcnc, ara h , lor which
thcrc is sumcicnt gcnomic scqucncc. Gcncspccinc primcr scts havc bccn dcsigncd lor T!LL!NG so
that mutations in cach copy ol ara h can bc scrccncd scparatcly. Tird, charactcrization ol allcrgcn gcnc
scqucncc has also allowcd thc isolation ol promotcrs that may bc usclul lor antilungal gcnc cxprcssion,
particularly whcn cxprcssion is to bc targctcd to thc dcvcloping sccd. Tis work is cxpcctcd to yicld, among
othcr things, promotcr scqucnccs that will bc usclul in achicving tissucspccinc cxprcssion ol dclcnsivc
transgcncs in dcvcloping pcanut sccds.
Sincc Aspergillus cntcrs thc maizc plant through thc silks, (Pccthambaran) bclicvcs it might bc possiblc
to inhibit inlcction by idcntilying silkprotcin markcrs associatcd with rcsistancc. Tus, thc protcomc
ol silks lrom rcsistant plants is bcing comparcd with that lrom susccptiblc oncs to attcmpt to idcntily
protcins produccd by nativc gcncs that arc involvcd in rcsistancc, and/or to idcntily protcin markcrs that
can bc uscd in a markcrassistcd brccding program. Protcins prcscnt in rcsistant lincs havc bccn idcntincd
and charactcrizcd, and havc bccn succcsslully tcstcd against A. favus in vitro.
Rcsistanccassociatcd protcins (RAPs) havc bccn idcntincd using protcomics to comparc constitutivc
protcin pronlcs bctwccn rcsistant and susccptiblc maizc gcnotypcs (Chcn). Translormcd corn plants wcrc
subscqucntly produccd in which silcncing ol thc targct RAPs was obscrvcd in somc lincs, and a kcrncl
scrccning assay dcmonstratcd a signincant incrcasc in susccptibility to A. favus colonization and aatoxin
production in thcsc lincs. Tcsc obscrvations arc consistcnt with thc hypothcsis that RAPs arc dircctly
involvcd in aatoxin rcsistancc in maizc, and thcrclorc gcncs cncoding thcsc protcins would bc cxccllcnt
candidatcs lor usc as molccular markcrs ol rcsistant corn lincs.
Tc production and tcsting ol transgcnic pcanut lincs cxprcssing an activc lorm ol thc maizc ribosomc
inactivating protcin, R!P ! was rcportcd (Vcissingcr). Pcanuts cxprcssing thc activc lorm ol thc R!P,
Mod ! wcrc tcstcd in vitro prcviously and havc bccn shown to bc rcsistant to Aspergillus inlcction All
ol thc lincs lound to bc rcsistant to A. favus wcrc subscqucntly tcstcd lor rcsistancc against two lcal
pathogcns, Sclerotinia minor and Sclerotium rolfsii, using a dctachcdlcal tcst that pcrmits quantincation ol
lungal growth. Four transgcnic lincs dcrivcd lrom thc runncr typc pcanut, Gcorgia Grccn, and onc linc
dcrivcd lrom thc \irginia cultivar, NC\ !, wcrc lound to cxhibit signincant rcsistancc comparcd with un
translormcd pcanut lincs. Tcsc lincs arc now bcing tcstcd against a rangc ol othcr lungal pathogcns, and
will also bc tcstcd to dctcrminc thc cxtcnt to which thc cnhanccd rcsistancc against A. favus inlcction is
rccctcd in rcduction ol aatoxin contamination.
Tcsting continucs ol lcrtilc, transgcnic cotton plants cxprcssing thc synthctic antimicrobial pcptidc,
4! (Rajasckaran). Transgcnic lincs produccd through Agrobacteriummcdiatcd translormation
(Rajasckaran ct al. Plant Biotechnology Journal 1: 44. 200) cxprcsscd thc antilungal gcnc product, and
in vitro assays ol plant lcal cxtracts connrmcd that 4! was cxprcsscd at sumcicnt lcvcls to inhibit thc
growth ol Fusarium verticillioides and Verticillium dahliae. Although in vitro assays did not show control ol
prcgcrminatcd sporcs ol Aspergillus favus, bioassays with cotton sccds in situ or in planta, inoculatcd with
a GFPcxprcssing A. favus, indicatcd that thc transgcnic cotton sccds inhibitcd cxtcnsivc colonization
and sprcad by thc lungus in cotylcdons and sccd coats. Transgcnic T! sccdlings had signincantly rcduccd
discasc symptoms and incrcascd sccdling lrcsh wcight, and thus tolcrancc to thc black root rot lungal
pathogcn ol cotton, ielaviopsis basicola. Ficld cvaluation ol T2 progcny lor rcsistancc against Fusarium
wilt racc ! indicatcd that thc transgcnic cntrics had improvcd stand, up to 68, comparcd with un
translormcd controls at 41.
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Crop Management
!nscct control is a major locus ol crop managcmcnt. Past studics in multiplc locations havc indicatcd that
signincant rcductions in lumonisins can occur on many corn hybrids cxprcssing thc 8t gcnc comparcd
to non8t hybrids whcn thc targct inscct (uropcan corn borcr) is controllcd at lcvcls approaching !00.
Howcvcr, this is only onc ol many dicrcnt inscct spccics that can promotc mycotoxins in thc various
susccptiblc crops. Fall armyworm lcal lccding and root knot ncmatodc damagc havc bccn invcstigatcd
lor thcir importancc in contributing to thc aatoxin problcm in corn in thc southcast (Krakowsky ct al.).
Root knot ncmatodcs appcar to also promotc aatoxin in pcanuts by cnhancing thc strcss duc to drought,
but may also contributc through physical damagc to pods (Timpcr ct al.). Codling moths, which promotc
aatoxin lormation in walnuts, havc bccn ccctivcly controllcd through usc ol a pcardcrivcd attractant
that can bc applicd in a microcncapsulatcd lorm in combination with rcduccd ratcs ol insccticidcs (Light).
Tc activity ol plant dcrivcd anthocyanins ( Johnson), chitinasc likc cnzymcs, pcroxidascs, and corn R!P
cnzymc (owd) studicd in dictary assays and transgcnically in plants, indicatc thcy havc thc potcntial to
bc variously combincd into stablc, broad spcctrum inscct rcsistancc through introductions ol compatiblc
gcncs.
Managcmcnt tactics in addition to inscct control, cspccially whcn conditions lavorablc lor mycotoxin
lormation can bc idcntincd, arc also ol potcntial usc. 8ccausc aatoxin can bc dcgradcd by ammonia,
and bccausc ammonia production is cnhanccd in plants trcatcd with Libcrty hcrbicidc, thcrc is potcntial
that cithcr LibcrtyLink plants (which dcgradc thc ammonia) or normal plants trcatcd with rclativcly
nontoxic lcvcls ol thc hcrbicidc, may havc rcduccd lcvcls ol aatoxin. nc study (8runs and Abbas) has
rcportcd no signincant rcduction ol aatoxin in corn in Mississippi, but anothcr study indicatcd that low
lcvcls ol Libcrty could signincantly rcducc aatoxin lcvcls whcn applicd sooncr (v. latcr) altcr mid silk by
ground to non LibcrtyLink hybrids (Moorc). Furthcr validation ol thc rcsults undcr thc samc timing
and application ratcs uscd in thc Louisiana study and additional optimization may lcad to an additional
control stratcgy lor aatoxin in corn.
Prcdicting whcn conditions lavorablc lor mycotoxin lormation occur is kcy to dcvcloping and
implcmcnting managcmcnt tactics. Rcmotc scnsing ol pcanut canopy indicatcd that it was ablc to givc
morc spccinc and timcly cstimatcs ol gcnotypc rcsponsc to drought than visual obscrvations (Sullivan).
Tis tcchniquc could bc uscd to cnhancc brccding progrcss ol drought and aatoxin rcsistant pcanut
varictics. A prcdictivc computcr program lor mycotoxin occurrcncc in Midwcst corn initially prcdictcd
that Aspergillus favus inoculum was likcly to bc prcscnt at problcm lcvcls at corn silking, and subscqucnt
prcdictions indicatcd that low lcvcls ol aatoxin wcrc likcly to bc prcscnt in corn at harvcst in ccntral
!llinois in 200 (owd ct al.). Grain clcvators in thc arca did rcjcct loads ol aatoxin contaminatcd corn
sporadically through ctobcr 200. Vidcr distribution ol this program, which has also givcn rcliablc
prcdictions ol lumonisin lcvcls ovcr thc past ycars, is likcly to promotc bcttcr managcmcnt ol mycotoxins
in corn.
Microbial Ecology
Considcrablc progrcss has bccn madc in thc usc ol compctitivc nonaatoxigcnic strains ol A. favus lor
rcducing aatoxin contamination in pcanuts in thc southcastcrn Unitcd Statcs and cottonsccd in Arizona
and Tcxas. 8iocontrol agcnts in thc lorm ol sporccoatcd barlcy (pcanuts) or colonizcd whcat (cotton)
rcccntly havc bccn commcrcially applicd to largc arcas ol crops. Aaguard
, thc biocontrol product

lor pcanuts, changcd soil populations lrom 7! to 4 toxigcnic strains ol A. favus and this ultimatcly
rcsultcd in an ovcrall aatoxin rcduction ol 8 in pcanuts (orncr). 8iocontrol application in pcanut
nclds providcs an additional bcncnt by controlling aatoxin contamination during pcanut storagc undcr
suboptimal tcmpcraturc and moisturc conditions. 8iological control in cotton may also havc a carryovcr
ccct in rcducing aatoxin contamination ol corn, a common rotation crop lor cotton (Cotty). Tc
primary locus now is to optimizc thc cost ol producing inoculum and to idcntily cnvironmcntal lactors
in thc ncld most conducivc to rcduction ol aatoxins. 8oth soil tcxturc and canopy shading arc critical lor
sporulation ol nonaatoxigcnic A. favus on whcat (Antilla and Cotty). Qucstionnaircs arc bcing providcd
to cotton larmcrs using biological control to bcttcr corrclatc spccinc cultivation practiccs with ccctivc
rcduction ol aatoxins.
8iocontrol tcchnology using nonaatoxigcnic A. favus strains also is bcing tcstcd with pistachios and
ngs in Calilornia (ostcr ct al.). 8iocontrol A. favus strains applicd to pistachio orchards pcrsistcd in
18
19
soil lor at lcast two ycars and continucd to displacc nativc toxigcnic strains (8097 ol isolatcs). qually
important, application ol A. favus AF16 docs not appcar to incrcasc lungal dccay ol carly split nuts. A.
favus AF16 was most prcvalcnt in soils undcr drip lincs in dripirrigatcd ng orchards. As with pistachios,
biological control did not incrcasc thc incidcncc ol ng dccay. thcr biocontrol stratcgics that arc bcing
pursucd includc thc usc ol ycasts and bactcria lor controlling invasion ol trcc nuts by aatoxigcnic lungi.
Tc ycast Pichia anomala VRL076 rcduccd thc lrcqucncy ol A. favus colonization ol woundcd pistachio
nuts by up to !0lold (Hua). Furthcrmorc, P. anomala will grow at low watcr activitics that arc conducivc to
invasion by A. favus. 8actcrial populations associatcd with almond rcproductivc parts arc bcing cxamincd
lor strains that arc antagonistic to A. favus (Palumbo ct al.).
Rcscarch in biological control tcchnology has providcd a wcalth ol knowlcdgc conccrning thc gcnctics
and dynamics ol natural populations ol aatoxigcnic lungi and how thcsc populations intcract with applicd
biocontrol strains. Scvcral lincs ol basic rcscarch arc bcing conductcd to bcttcr undcrstand thc mcchanisms
undcrlying thc inhibition and cxclusion ol toxigcnic lungi lrom crops by biocontrol agcnts. A laboratory
assay in which woundcd viablc pcanut sccds arc inoculatcd with soil lrom thc ncld indicatcs that scction
Flavi spccics prclcrcntially invadc pcanuts at 2217 C and 0.920.96 sccd watcr activity (Horn). !t also
appcars that compctition within scction Flavi in natural populations accounts lor considcrablc rcduction in
aatoxin contamination. thcr rcscarch suggcsts that chitinasc production is onc mcchanism undcrlying
thc parasitism ol Aspergillus and Fusarium spccics by Gliocladium catenulatum (Kcndra ct al.).
Natural Products for Prevention of Fungal Invasion and/or Aflatoxin Biosynthesis
Considcrablc advanccs havc bccn madc in thc idcntincation ol naturallyoccurring compounds in both
corn and trcc nuts that supprcss thc lormation ol aatoxins. Two compounds (A8!! and A8!2) havc
bccn idcntincd in kcrncls ol thc rcsistant maizc, Tcx6, that supprcss both lungal growth and aatoxin
biosynthcsis (Paync). A8!2 was a lcss potcnt inhibitor ol lungal growth than A8!! but had morc ccct
on aatoxin biosynthcsis. 8oth compounds appcar to supprcss transcription ol pathway gcncs, but act
dicrcntly on othcr rcgulatory gcncs. !nitial structural charactcrization showcd thcsc compounds to bc
nonprotcinaccous, hcat labilc, small molcculcs, whilc thc mass spcctromctric molccular wcight suggcstcd
thc prcscncc ol a nitrogcn atom. Tc inhibitors appcar to bclong to thc inositol polyphosphatc class,
rclatcd to phytic acid, although thc lattcr compound showcd no inhibitory activity.
Prcvious work on polyphcnolic constitucnts ol walnut that inhibit aatoxin biosynthcsis has bccn
cxtcndcd to othcr phcnolic antioxidants prcscnt in almonds and pistachios (Molyncux). Tcsc includcd
hydrolysablc tannins, thc avonoid, catcchin, and a scrics ol phcnolic acids common to trcc nuts and
othcr plant spccics. Tc commcrcial antioxidant, lauryl gallatc, was also tcstcd as a modcl lor thc anacardic
acids prcscnt in pistachio hulls. Tc most potcnt compounds wcrc pcntagalloyl glucosc, cacic acid and
lauryl gallatc, cach ol which inhibitcd aatoxin production by ~99. Tcsting in thc prcscncc and abscncc
ol pcroxidc showcd that phcnolic compounds wcrc ablc to ovcrcomc aatoxin production induccd by
oxidativc strcss and this was connrmcd using singular gcnc dclction mutants ol Saccharomyces cerevisiae as
a modcl lungal systcm to cxaminc lunctional gcnomics ol oxidativc strcss rcsponscs. Tc rcsults indicatc
that aatoxin production is stimulatcd by oxidativc strcss and that phcnolic compounds prcscnt in trcc
nuts arc capablc ol supprcssing aatoxin biosynthcsis, implying that brccding to cnhancc lcvcls ol natural
phcnolics should rcducc thc potcntial lor aatoxin contamination.
Dr. Jane F. Robens
National Program Leader
Food Safety and Health
Beltsville Agricultural Research Center
Agricultural Research Service, USDA
Beltsville, MD
18
19
AGENDA
5
th
Annual Fungal Genomics,
6
th
Annual Fumonisin,
18
th
Annual Afatoxin Elimination Workshop
October 24-26, 2005
Marriott Crabtree Valley Raleigh, NC
SUNDAY, OCTOBER 23, 2005
3:00 - 6:00 REGISTRATION / POSTER ASSIGNMENTS
MONDAY, OCTOBER 24, 2005
7:15 Load buses for travel to Cotton Incorporated World Headquarters (Cary, NC)
8:00 Doughnuts, Coffee, and Presentations and Tour of Cotton Incorporated
11:30 Depart for Bayer CropScience (Research Triangle Park, NC)
12:00 Lunch and Tour of Bayer CropScience (Gustafson) Research Labs
2:30 Depart for NC State University (Raleigh, NC)
3:30 Tour NC State University Centennial Campus Research Facilities
5:00 Return to Marriott Crabtree Valley
Dinner on your own
4:00 7:00 REGISTRATION / POSTER ASSIGNMENTS
20
21
TUESDAY, OCTOBER 25, 2005
7:00 8:00 CONTINENTAL BREAKFAST
8:00 Welcome
Roy Cantrell, Cotton Incorporated
8:05 Introductory Remarks
Jane F. Robens, USDA-ARS, National Program Leader, Beltsville, MD
5
TH
ANNUAL FUNGAL GENOMICS WORKSHOP
MODERATOR: Roy Cantrell, Cotton Incorporated
8:10 Finding Target Genes for Better Control of Aspergillus. Jong H. Kim
1
, Bruce
C. Campbell
1
, Jiujiang Yu
2
, Gregory S. May
3
, Kathleen L. Chan
1
, Gary A. Payne
4
,
Deepak Bhatnagar
2
, and Thomas E. Cleveland
2
.
1
USDA-ARS, Western Regional
Research Center, Albany, CA;
2
USDA-ARS, Southern Regional Research Center,
New Orleans, LA;
3
MD Anderson Cancer Center, University of Texas, Houston, TX;
4
Department of Plant Pathology, North Carolina State University, Raleigh, NC.
8:25 Comparative Genomic Analysis of Secondary Metabolite Gene Clusters of
Closely Related Aspergilli. William C. Nierman
1
, Natalie D. Fedorova
1
, Catherine
M. Ronning
1
, Jennifer Wortman
1
, Masayuki Mashida
2
, Jiujiang Yu
3
, Thomas E.
Cleveland
3
, Deepak Bhatnagar
3
, and Gary A. Payne
4
.
1
The Institute for Genomic
Research, Rockville, MD;
2
Institute for Biological Resources and Functions, Nat.
Inst. Of Advanced Ind. Sci. and Technol., Tsukuba, Japan;
3
USDA-ARS, Southern
Regional Research Center, New Orleans, LA;
4
Department of Plant Pathology, North
Carolina State University, Raleigh, NC.
8:40 Aspergillus favus Genomics in Discovering Genes Involved in Afatoxin
Biosynthesis. Jiujiang Yu
1
, Jeffery R. Wilkinson
1
, William C. Nierman
2
, H. Stanley
Kim
2
, Gary A. Payne
3
, Bruce C. Campbell
4
, Deepak Bhatnagar
1
, and Thomas E.
Cleveland
1
.
1
USDA-ARS, Southern Regional Research Center, New Orleans,
LA;
2
The Institute for Genomic Research, Rockville, MD;
3
Department of Plant
Pathology, North Carolina State University, Raleigh, NC;
4
USDA-ARS, Western
Regional Research Center, Albany, CA.
8:55 Mining Expressed Sequence Tags (ESTs) Leads to Identifcation of Putative
FUM Cluster Transcription Factor. Daren W. Brown, Robert A. E. Butchko, Mark
Busman, and Robert H. Proctor. USDA-ARS, National Center for Agricultural
Utilization Research, Peoria, IL.
20
21
9:10 Release of the Aspergillus favus Genome Sequence. Gary A. Payne
1
, B.
Pritchard
1
, Jiujiang Yu
2
, William C. Nierman
3
, Ralph Dean
1
, Deepak Bhatnagar
2
,
and Thomas E. Cleveland
2
.
1
Department of Plant Pathology, North Carolina State
University, Raleigh, NC;
2
USDA-ARS, Southern Regional Research Center, New
Orleans, LA;
3
The Institute for Genomic Research, Rockville, MD.
9:25 Production of Cyclopiazonic Acid, Afatrem, and Afatoxin is Regulated by
veA, a Gene Necessary for Sclerotial Formation in Aspergillus favus. Rocio M.
Duran
1
, Jeffrey W. Cary
2
, and Ana M. Calvo
1
.
1
Department of Biological Sciences,
Northern Illinois University, Dekalb, IL;
2
USDA-ARS, Southern Regional Research
Center, New Orleans, LA.
9:40 10:00 PANEL DISCUSSION
Panel Chair: Gary A. Payne, North Carolina State University, Raleigh, NC

10:00 10:20 BREAK AND POSTER VIEWING
6
TH
MODERATOR: Larry Antilla, Arizona Cotton Research and Protection Council
10:20 Kernel Constituents Induce Fumonisin Production during Colonization by
Fusarium verticillioides. Charles Woloshuk, Purdue University, West Lafayette, IN.
10:35 Genetics and Breeding of Host Resistance to Fusarium Ear Rot and Fumonisin
Contamination. James Holland. Department of Crop Science, North Carolina State
University, Raleigh, NC.
10:50 NIR Spectroscopy as a Tool for Optimizing Sorting of White Corn Kernels
Contaminated with Fumonisin. Tom C. Pearson
1
and Donald T. Wicklow
2
.
1
USDA-ARS, Grain Marketing and Production Research Center, Manhattan, KS;
2
USDA-ARS, National Center for Agricultural Utilization Research, Peoria, IL.
11:05 Maize LOX3 Gene is Required for Fumonisin Biosynthesis and Conidiation
of Fusarium verticillioides. Xiquan Gao
1
, Won-Bo Shim
1
, Ivo Feussner
2
, and
Mike Kolomiets
1
.
1
Department of Plant Pathology and Microbiology, Texas
A&M University, College Station, TX;
2
Georg-August University of Gettingen,
Gettingen, Germany.
11:20 Toxicity Responses of Corn to the Mycotoxin Fumonisin B
1
in the Absence
of Fusarium verticillioides Infection. Anne Marie Zimeri, Lonnie D. Williams,
Ronald T. Riley, and Anthony E. Glenn. USDA-ARS, Richard B. Russell Research
Center, Athens, GA.
22
23
Panel Chair: Charles Woloshuk, Purdue University, West Lafayette, IN
12:00 1:00 LUNCH
18
TH
Session 1: Crop Resistance Conventional Breeding
MODERATOR: Don Jones, Cotton Incorporated
1:00 Progress on the Creation of Usable Commercial Inbreds and Hybrids with Low
Afatoxin in Grain Using Molecular Markers. Don White and Torbert Rocheford,
University of Illinois, Urbana, IL.
1:15 Breeding Corn Germplasm for Agronomic Performance and Reduced Afatoxin
Contamination. Javier Betrn
1
, Tom Isakeit
2
, Gary Odvody
3
, and Kerry Mayfeld
1
.
1
Soil & Crop Sciences Department, Texas A&M University, College Station, TX;
2
Department of Plant Pathology and Microbiology, Texas A&M University, College
Station, TX;
3
Texas A&M Research & Extension Center, Corpus Christi, TX.
1:30 Interaction Between Aspergillus favus Strains and Host Plant Genotypes
Across Environments and Years. Kerry Mayfeld
1
, Tom Isakeit
2
, Gary Odvody
3
,
and Javier Betrn
1
.
1
Soil & Crop Sciences Department, Texas A&M University,
College Station, TX;
2
Department of Plant Pathology and Microbiology, Texas A&M
University, College Station, TX;
3
Texas A&M Research & Extension Center, Corpus
Christi, TX.
1:45 Application of HACCP to Control Mycotoxins in Maize Breeding Programs.
David F. Kendra. USDA-ARS, National Center for Agricultural Utilization
Research, Peoria, IL.
2:00 Suppression of Insect Mediated Afatoxin Contamination of Almond. T.
M. Gradziel and A. H. Dandekar. Department of Plant Sciences, University of
California at Davis, Davis, CA.
2:15 Genetic and Genomic Approaches to Improve Host Resistance to Preharvest
Afatoxin Contamination in Corn and Peanut. Baozhu Guo
1
, M. Luo
2
, H.
Chen
3
, A. E. Coy
2
, Matthew D. Krakowsky
4
, C. Corley Holbrook
4
, X. Liang
5
, R.
Dewey Lee
2
, and Craig K. Kvien
3
.
1
USDA-ARS, Crop Protection and Management
Research Unit, Tifton, GA;
2
Department of Crop and Soil Sciences, University of
Georgia, Tifton, GA;
3
NESPAL, University of Georgia, Tifton, GA;
4
USDA-ARS,
Crop Genetics and Breeding Research Unit, Tifton, GA;
5
Guangdong Academy of
Agricultural Sciences, Guangzhou, China.
22
23
2:50 Progress Toward Identifying New Sources of Genetic Variation Associated with
Reduced Levels of Afatoxin Accumulation in Maize. Thomas Brooks
1
, Matthew
Krakowsky
2
, W. Paul Williams
1
, and Gary Windham
1
.
1
USDA-ARS, Corn Host
Plant Resistance Research Unit, Mississippi State, MS;
2
USDA-ARS, Crop Genetics
and Breeding Research Unit, Tifton, GA.
3:05 Proteomic Identifcation of Maize Cob Proteins that Potentially Confer
Resistance to Afatoxin. Dawn S. Luthe
1
, Olga Pechanova
1
, Bele Peethambaran
1
,
Leigh Hawkins
2
, Tibor Pechan
3
, Gary Windham
4
, Susan Bridges
5
, and W. Paul
Williams
4
.
1
Department of Biochemistry and Molecular Biology, Mississippi
State University, Mississippi State, MS;
2
USDA-ARS, Corn Host Plant Resistance
Research Unit, Mississippi State, MS;
3
Life Sciences and Biotechnology Institute,
Mississippi State University, Mississippi State, MS;
4
USDA-ARS, Corn Host Plant
Resistance Research Unit, Mississippi State, MS;
5
Department of Computer Science,
Mississippi State University, Mississippi State, MS.
3:20 A Field Technique for Varietal Assessment of Second Phase Afatoxin
Contamination in Cotton. Mary W. Olsen
1
and Peter J. Cotty
2
.
1
Division of Plant
Pathology and Microbiology, University of Arizona, Tucson, AZ;
2
USDA-ARS,
Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ.
3:35 Corn Hybrids with Exotic Germplasm and Low Afatoxin. Wenwei Xu
1
, Gary
Odvody
2
, and W. Paul Williams
3
.
1
Agricultural Research and Extension Center,
Texas A&M University, Lubbock, TX;
2
Agricultural Research and Extension
Center, Texas A&M University, Corpus Christi, TX;
3
Resistance Research Unit, Mississippi State, MS.
3:50 Computational Tools for Protein Identifcation and Gene Ontology Annotation
of the Maize Proteome. Susan M. Bridges
1
, Julia E. Hodges
1
, Gregory Bryce
Magee
1
, Nan Wang
1
, Dawn S. Luthe
2
3
.
1
Department of
Computer Science and Engineering, Mississippi State University, Mississippi
State, MS;
2
Department of Biochemistry and Molecular Biology, Mississippi
State University, Mississippi State, MS;
3
Research Unit, Mississippi State, MS.
4:05 Progress in Breeding Peanut for Resistance to Preharvest Afatoxin
Contamination and Drought. C. Corley Holbrook
1
, Baozhu Guo
2
, David M.
Wilson
3
, Dana G. Sullivan
4
, Emily Cantonwine
3
, and Craig K. Kvien
5
.
1
USDA-
ARS, Crop Genetics and Breeding Research Unit, Tifton, GA;
2
USDA-ARS, Crop
Protection and Management Research Unit, Tifton, GA;
3
Department of Plant
Pathology, University of Georgia, Tifton, GA;
4
USDA-ARS, Southeast Watershed
Research Laboratory, Tifton, GA;
5
NESPAL, University of Georgia, Tifton, GA.
24
25
4:20 Searching for New Resistance and Control Measures of Afatoxin in Corn.
Steven Moore
1
, Hamed Abbas
2
, and Mark Millard
3
.
1
Louisiana State University
Agricultural Center, Alexandria, LA;
2
USDA-ARS, Crop Genetics and Production
Research Unit, Stoneville, MS.
3
North Central Regional Plant Introduction Station,
Ames, IA.
4:35 Development of Afatoxin-resistant Maize Inbreds and Identifcation of
Potential Resistance Markers through USA-Africa Collaborative Research.
Robert L. Brown
1
, Zhi-Yuan Chen
2
, Abebe Menkir
3
, Ranajit Bandyopadhyay
3
, and
Thomas E. Cleveland
1
.
1
USDA-ARS, Southern Regional Research Center, New
Orleans, LA;
2
Department of Plant Pathology and Crop Physiology, Louisiana
State University, Baton Rouge, LA;
3
International Institute of Tropical Agriculture,
Ibadan, Nigeria.
Panel Chair: Don White, University of Illinois, Urbana, IL

6:00 7:30 POSTER VIEWING WITH HORS DOUVRES AND BEVERAGES
WEDNESDAY, OCTOBER 26, 2005
7:00 8:00 CONTINENTAL BREAKFAST

Session 2: Microbial Ecology
MODERATOR: Phil Wakelyn, National Cotton Council
8:00 Effect of Fungal Competition on the Colonization of Wounded Peanut Seeds by
Aspergillus Section Flavi from Natural Soil Populations. Bruce W. Horn, USDA-
ARS, National Peanut Research Laboratory, Dawson, GA.
8:15 Transfer of Afatoxin Biocontrol Technology: Results of First Commercial Use
in Peanuts. Joe W. Dorner. USDA-ARS, National Peanut Research Laboratory,
Dawson, GA.
8:30 Atoxigenic Strain Technology for Afatoxin Control in Cotton. Larry Antilla,
Arizona Cotton Research and Protection Council, Phoenix, AZ.
8:45 Managing Afatoxins in Cotton-Corn Rotations. Peter J. Cotty, USDA-ARS,
Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ.
24
25
9:00 Afatoxin Control in Pistachios: Biocontrol Using Atoxigenic Strains. Themis
Michailides and Mark Doster. University of California, Davis/Kearney Agricultural
Center, Parlier, CA.
9:15 Afatoxin Control in Figs: Biocontrol and New Resistant Cultivars. Mark Doster
and Themis Michailides. University of California, Davis/Kearney Agricultural
Center, Parlier, CA.
9:30 Identifcation of Bacterial Antagonists of Aspergillus favus from California
Almond Orchards. Jeffrey D. Palumbo, James L. Baker, and Noreen Mahoney.
USDA-ARS, Western Regional Research Center, Albany, CA.
9:45 Biological Control of Aspergillus favus by a Saprophytic Yeast Strain in Tree-
nut Orchards: Progress in 2005. Sui Sheng Hua. USDA-ARS, Western Regional
Research Center, Albany, CA.
Panel Chair: Bruce W. Horn, USDA-ARS, National Peanut Research
Laboratory, Dawson, GA
Session 3: Crop Resistance Genetic Engineering
MODERATOR: Keerti Rathore, Texas A&M University
10:40 Genetic Engineering of Peanut with Putative Antifungal Genes. Y. Chu
1
, P.
Faustinelli
1
, L. Ramos
1
, K. Rajasekaran
2
, J. Cary
2
, and P. Ozias-Akins
1
.
1
Department
of Horticulture and NESPAL, University of Georgia Tifton Campus, Tifton, GA;
2
USDA-ARS, Southern Regional Research Center, New Orleans, LA.
10:55 Transgenic Peanuts with Enhanced Resistance to Aspergillus favus. Arthur K.
Weissinger, Department of Crop Science, North Carolina State University, Raleigh,
NC.
11:10 Identifcation, Characterization and Antifungal Activities of Silk Proteins in
Aspergillus favus Resistant and Susceptible Corn Inbreds. Bela Peethambaran
1
,
Gary L. Windham
2
, Leigh Hawkins
2
, W. Paul Williams
2
, and Dawn S. Luthe
1
.
1
Department of Biochemistry and Molecular Biology, Mississippi State University,
Mississippi State, MS;
2
USDA-ARS, Corn Host Plant Resistance Research Unit,
Mississippi State, MS.
26
27
11:25 Silencing the Expression of RAP Genes in Maize and the Effect on Host
Resistance against Aspergillus favus Infection and Afatoxin Production. Zhi-
Yuan Chen
1
, Robert L. Brown
2
, Thomas E. Cleveland
2
, and Kenneth E. Damann
1
.
1
Department of Plant Pathology and Crop Physiology, Louisiana State University
Agricultural Center, Baton Rouge, LA;
2
Center, New Orleans, LA.
11:40 Genetic Engineering of Cotton for Phytopathogens Including Aspergillus
favus. Kanniah Rajasekaran
1
, Jeffrey W. Cary
1
, and Mauricio Ulloa
2
.
1
USDA-
ARS, Southern Regional Research Center, New Orleans, LA;
2
USDA-ARS, Western
Integrated Cropping Systems Research Unit, Shafter, CA.
12:00 1:00 LUNCH
Panel Chair: Arthur K. Weissinger, North Carolina State University,
Raleigh, NC
Session 4: Crop Management and Handling, Insect Control, and Fungal Relationships
MODERATOR: Pat OLeary, Cotton Incorporated
1:20 Update on Validation and Distribution of a Computer Program for Predicting
Mycotoxins in Midwest Corn. Patrick F. Dowd. USDA-ARS, National Center for
Agricultural Utilization Research, Peoria, IL.
1:35 Mechanism of Preharvest Afatoxin Contamination in Peanut Infected by Root-
Knot Nematodes. Patricia Timper
1
, C. Corley Holbrook
2
, and David M. Wilson
3
.
1
USDA-ARS, Crop Protection and Management Research, Tifton, GA;
2
USDA-
ARS, Crop Genetics and Breeding Research Unit, Tifton, GA;
3
Department of Plant
Pathology, University of Georgia, Tifton, GA.
1:50 Experimental Use of the Pear Ester Kairomone to Improve Codling Moth
Control in Walnuts. Douglas M. Light, Paula I. Bouyssounouse, and Bruce C.
Campbell. USDA-ARS, Western Regional Research Center, Albany, CA.
2:05 Cultural Conditions Promoting Chitinase Production by Gliocladium
catenulatum. David F. Kendra
1
, Michael J. Muhitch
2
, Amber Anderson
1
, and
Cesaria E. McAlpin
1
.
1
USDA-ARS, National Center for Agricultural Utilization
Research, Peoria, IL;
2
Rochester College, Rochester Hills, MI.
2:20 Liberty Link and Urea on Afatoxin and Fumonisin Levels in Corn. H. Arnold
Bruns and H. K. Abbas. USDA-ARS, Crop Genetics and Production Research Unit,
Stoneville, MS.
26
27
Panel Chair: David F. Kendra, USDA-ARS, National Center for
Agricultural Utilization Research, Peoria, IL
Session 5: Detection, Extraction, and Analysis of Afatoxins; Potential Use of Natural
Products for Prevention of Fungal Invasion and/or Afatoxin Biosynthesis in Crops
MODERATOR: Tom Wedegaertner, Cotton Incorporated
3:15 Distribution of Afatoxin in Non-irrigated Peanuts. Thomas F. Schatzki and M.
S. Ong. USDA-ARS, Western Regional Research Center, Albany, CA.
3:30 Inhibition of Aspergillus favus Afatoxin Biosynthesis by Antioxidant
Phytochemicals Occurring in Tree Nuts. Russell J. Molyneux, Noreen Mahoney,
Bruce C. Campbell, and Jong H. Kim. USDA-ARS, Western Regional Research
Center, Albany, CA.
3:45 Biochemical and Genetic Analysis of Gallic Acid in Walnuts in Relation to
Afatoxin Accumulation. Ryann M. Muir
1
, Elizabeth Ingham
1
, Sandra Uratsu
1
,
Gale McGranahan
1
, Charles Leslie
1
, Noreen Mahoney
2
, and Abhaya Dandekar
1
.
1
Department of Pomology, University of California, Davis, CA;
2
USDA-ARS,
Western Regional Research Center, Albany, CA.
4:00 Inhibition of Afatoxin Production by Compounds in Corn Seeds. Gary
A. Payne
1
, Robert A. Holmes
2
, and Rebecca S. Boston
2
.
1
Department of Plant
Pathology, North Carolina State University, Raleigh, NC;
2
Department of Botany,
North Carolina State University, Raleigh, NC.
Panel Chair: Russell J. Molyneux, USDA-ARS, Western Regional
Research Center, Albany, CA.

4:35 5:15 COMMODITY BREAKOUT SESSIONS

6:00 7:00 RECEPTION

7:00 BANQUET
28
29
A. Fungal Genomics, Regulation of Afatoxin Biosynthesis
A-1 Evolutionary processes in the afatoxin gene cluster in Aspergillus. I.
Carbone
1
, J. L. Jakobek
1
, E. H. Moussa
1,2
, J. E. Cox
1
, and B. W. Horn
3
.
1
Center for Integrated Fungal Research, Department of Plant Pathology, North
Carolina State University, Raleigh, NC;
2
Present address: Faculty of Sciences
II, Department of Biological Sciences, Lebanese University, Beirut, Lebanon;
3USDA-ARS, National Peanut Research Laboratory, Dawson, GA.
A-2 Differential Gene Expression Levels for Aspergillus favus Resistance in
Two Inbred Maize Lines. Rowena Y. Kelley
1
, Deborah L. Boykin
2
, Leigh
K. Hawkins
3
3
.
1
Department of Plant and Soil Sciences,
Mississippi State University, Mississippi State, MS;
2
USDA-ARS, Mid
South Area Statistics Offce, Stoneville, MS;
3
Resistance Research Unit, Mississippi State, MS.
A-3 Enhanced Activity of Fungicides by Positive Interaction with Natural
Phenolic Agents; Target-gene Based Bioassays for Control of Aspergilli.
Jong H. Kim
1
, Bruce C. Campbell
1
, Jiujiang Yu
2
, Noreen Mahoney
1
, Kathleen
L. Chan
1
, Russell J. Molyneux
1
, Deepak Bhatnagar
2
, Thomas E. Cleveland
2
,
Gregory S. May
3
, and Gary A. Payne
4
.
1
USDA-ARS, Western Regional
Research Center, Albany, CA;
2
Center, New Orleans, LA;
3
MD Anderson Cancer Center, University of Texas,
Houston, TX;
4
Department of Plant Pathology, North Carolina State University,
Raleigh, NC.
A-4 Deletion of GBP1, a Gene Encoding a Monomeric G Protein, De-represses
Fumonisin Biosynthesis in Fusarium verticillioides. Uma Sagaram and
Won-Bo Shim. Department of Plant Pathology and Microbiology, Texas A&M
University, College Station, TX.
A-5 A Link between Rho-Signaling and Afatoxin Biosynthesis in Aspergillus
favus. D. Ryan Georgianna
1,2
, Michael S. Price
1,3
, and Gary A. Payne
1,2
.
1
Center for Integrated Fungal Research and Department of Plant Pathology,
North Carolina State University, Raleigh, NC;
2
Genomic Sciences Graduate
Program, North Carolina State University, Raleigh, NC;
3
Department of
Molecular Genetics and Microbiology, Duke University Medical Center,
Durham, NC.
A-6 nadA, a Gene Regulated by AfR, Does Not Appear to Affect Afatoxin
Production. Carrie Jacobus
1
, Gary A. Payne
2
, and Niki Robertson
1,3
.
1
Department of Genetics, North Carolina State University, Raleigh, NC;
2
Department of Plant Pathology, North Carolina State University, Raleigh, NC;
28
29
3
Department of Botany, North Carolina State University, Raleigh, NC.
A-7 Metabolic Profling of Aspergillus favus during Afatoxin Biosynthesis. N.
J. Glassbrook and G. A. Payne. Department of Plant Pathology, North Carolina
State University, Raleigh, NC.
B. Fumonisin Elimination
B-1 Mapping of QTL for Fusarium Ear Rot and Fumonisin Accumulation in
Maize. Leilani A. Robertson
1
, Michael P. Jines
2
, Peter Balint-Kurti
3
, Craig
E. Kleinschmidt
4
, Don G. White
4
, Gary A. Payne
5
, Chris M. Maragos
6
, and
James B. Holland
3
.
1
Deptartments of Plant Pathology and Crop Science, North
2
Department of Crop Science, North
3
USDA-ARS, Plant Science Research
Unit, Department of Crop Science, North Carolina State University, Raleigh,
NC;
4
Department of Crop Sciences, University of Illinois, Urbana-Champaign,
IL;
5
Department of Plant Pathology, North Carolina State University, Raleigh,
NC;
6
USDA-ARS, National Center for Agricultural Utilization Research,
Peoria, IL.
B-2 Polyketide Synthase in Fusarium verticillioides: Can They be Exploited to
Control Fumonisin Contamination in Corn? Robert H. Proctor, Robert A. E.
Butchko, Mark Busman, Anne E. Desjardins, Daren W. Brown, and Ronald D.
Plattner. USDA-ARS, National Center for Agricultural Utilization Research,
Peoria, IL.
B-3 Computational Studies on the Infuence of Solvent on the Conformational
Preferences and Selective Recognition of Fumonisins. M. Appell, C. M.
Maragos, and D. F. Kendra. USDA-ARS, National Center for Agricultural
Utilization Research, Peoria, IL.
B-4 Characterization of Potential Fumonisin Regulatory Genes Identifed by
EST Analysis. Robert A. E. Butchko, Robert H. Proctor, Daren Brown, and
Murk Busman. USDA-ARS, National Center for Agricultural Utilization
Research, Peoria, IL.
B-5 Fumonisins in Maize in Guatemala, Exposure Estimates, and Policies and
Recommendations to Minimize Exposure. Ronald T. Riley
1
, Olga A. Torres
2
,
Rubin Grajeda
2
, Edwin Palencia
2
, L. Lopez de Pratdesaba
2
, Anthony E. Glenn
1
,
Kerry ODonnell
3
, Mario Fuentes
4
, and Marcy Speer
5
.
1
USDA-ARS, Richard
B. Russell Research Center, Athens, GA;
2
Instituto de Nutricion de Centro
America Y Panama, Calzada Roosevelt, Zone 11, Guatemala;
3
USDA-ARS,
National Center for Agricultural Utilization Research, Peoria, IL;
4
Institute of
Agricultural Science and Technology, Guatemala;
5
Duke Center for Human
Genetics, Duke University, Durham, NC.
30
31
B-6 Fusaric Acid, a Fusarium verticillioides Miasma to Bacillus mojavensis,
a Biological Control Bacterial Endophyte. Charles W. Bacon and D. M.
Hinton. USDA-ARS, Richard B. Russell Research Center, Athens, GA.
B-7 Developmental Toxicity of Fusarium verticillioides and Fumonisin B
1

in LMBc and CD1 Mice: Comparing the in vivo Models. Kenneth A.
Voss
1
, Ronald T. Riley
1
, Tantiana D. Burns
1,2
and Janee B. Gelineau-van
Waes
3
.
1
USDA-ARS, Richard B. Russell Research Center, Athens, GA;
2
Interdisciplinary Toxicology Program, University of Georgia, Athens, GA;
3
Department of Genetics, Cell Biology, and Anatomy, Nebraska Medical
Center, Omaha, NE.
C. Afatoxin - Crop Management and Handling, Insect Control, and Fungal
Relationships
C-1 Anthocyanins from Petunia Floral Structures that Inhibit Corn Earworm
Development. Eric T. Johnson and Patrick F. Dowd. USDA-ARS, National
Center for Agricultural Utilization Research, Peoria, IL.
C-2 Using a Remotely Sensed Crop Index to Enhance Selection for Drought
Tolerant Peanuts. Dana G. Sullivan
1
and C. Corley Holbrook
2
.
1
USDA-ARS,
Southeast Watershed Research Laboratory, Tifton, GA;
2
USDA-ARS, Crop
Genetics Breeding and Research Unit, Tifton, GA.
C-3 Correlations between Biotic Stresses and Afatoxin Contamination
in Maize. Matthew Krakowsky
1
, Xinzhi Ni, Richard Davis, and Kedong
Da.
1
USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GA;
2
USDA-ARS, Crop Protection and Management Research Unit, Tifton, GA;
3
Department of Entomology, Coastal Plain Experiment Station, University of
Georgia, Tifton, GA.
D. Afatoxin - Crop Resistance Conventional Breeding
D-1 Multilocation Evaluation of Afatoxin Accumulation in Yellow Maize
Hybrids. Cody McKee
1
, Tom Isakeit
2
, Gary Odvody
3
, Kerry Mayfeld
1
, and
Javier Betrn
1
.
1
Soil & Crop Sciences Department, Texas A&M University,
2
Department of Plant Pathology and Microbiology, Texas
A&M University, College Station, TX;
3
Texas A&M Research & Extension
Center, Corpus Christi, TX.
D-2 Southeastern Regional Afatoxin Test (SERAT). Michael Clements
1
, Paul
Williams
1
, Steve Moore
2
, Matthew Krakowky
3
, Baozhu Guo
3
, Don White
4
,
Wenwei Xu
5
, Tom Isakeit
6
, Tom Brooks
1
, Gary Windham
1
, Hamed Abbas
7
,
James Perkins
8
, Daniel Gorman
9
, Quinton Raab
10
, Keith Arnold
10
, David
Smith
11
, and Javier Betrn
6
.
1
USDA-ARS, Mississippi State, MS;
2
Louisiana
30
31
State University Agricultural Center, Alexandria, LA;
3
USDA-ARS, Tifton,
GA;
4
University of Illinois, Urbana, IL;
5
Texas A&M University Agricultural
Research and Extension Center, Lubbock, TX;
6
Texas A&M University,
7
USDA-ARS, Stoneville, MS;
8
Monsanto Company Crop
Protection, Waterman, IL;
9
Pioneer Dupont, Cairo, GA;
10
B-H Genetics,
Moulton, TX;
11
Zea Sage, Sycamore, Il.
D-3 Response of Afatoxin of CIMMYT Germplasm in Southern USA. Dan
Jeffers
1
, Matthew Krakowsky
2
, Paul Williams
3
, and Javier Betrn
4
.
1
CIMMYT,
Mexico D. F.,
2
USDA-ARS, Tifton, GA;
3
4
Texas A&M University, College Station, TX.
D-4 Phenotypic and Genotypic Characterization of a RIL Maize Mapping
Population for Afatoxin and Secondary Traits. Melanie Edwards, Monica
Menz, Tom Isakeit, and Javier Betrn. Texas A&M University, College Station,
TX.
D-5 Expression of LOX Pathway Genes in Corn Embryos Associated with
Aspergillus favus Resistance. A. Camas
1
, L. Lopez
1
, G. Windham
2
, P.
Williams
2
, and D. S. Luthe
1
.
1
Department of Biochemistry and Molecular
Biology, Mississippi State University, Mississippi State, MS;
2
USDA-ARS,
Corn Host Plant Resistance Research Unit, Mississippi State, MS.
D-6 Breeding for Increased Resistance to Fusarium verticillioides in Maize.
Magen Starr
1
, Leilani Robertson
1
, James Holland
2
, and Gary Payne
3
.
1
Department of Crop Science, North Carolina State University, Raleigh, NC;
2
USDA-ARS, Department of Crop Science, North Carolina State University,
Raleigh, NC;
3
Raleigh, NC.
D-7 Quantitative Expression Analysis of Adversity Resistance Genes in Corn
Germplasm with Resistance to Preharvest Afatoxin Contamination. M.
Luo
1
, R. D. Lee
2
, and B. Z. Guo
2
.
1
Department of Crop and Soil Sciences,
University of Georgia, Tifton, GA;
2
USDA-ARS, Crop Protection and
Management Research Unit, Tifton, GA.
D-8 Peanut PR Protein, -1,3-glucanase, Induction by Aspergillus favus and
Copurifcation with a Conglutin-like Protein. X. Liang
1
, B. Z. Guo
2
, and C.
C. Holbrook
3
.
1
Guangdong Academy of Agricultural Sciences, Guangzhou,
China;
2
USDA-ARS, Crop Protection and Management Research Unit, Tifton,
GA;
3
USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GA.
D-9 Corn Husk Characteristics Potentially Associated with Resistance to
Afatoxin Contamination of Grain: A Preliminary Study. M. J. Clements
and W. P. Williams. USDA-ARS, Corn Host Plant Resistance Research Unit,
Mississippi State, MS.
32
33
D-10 Chalcone Synthase, a Gene that Infuences Both Drought Response and
Afatoxin Accumulation in Maize. M. Gerau, D. Bush, D. Davis, C. Morriss,
and G. Davis. Division of Plant Sciences, University of Missouri-Columbia,
Columbia, MO.
E. Afatoxin - Microbial Ecology
E-1 Infuences of Crops and Geographic Features on Communities of
Afatoxin-producing Fungi. Ramon Jaime
1
and Peter J. Cotty
2
.
1
Division of
Plant Pathology and Microbiology, University of Arizona, Tucson, AZ;
2
USDA-
ARS, Division of Plant Pathology and Microbiology, University of Arizona,
Tucson, AZ.
E-2 Afatoxin Contamination of Maize in Africa. Claudia Probst
1
, Henry
Njapau
2
, and Peter J. Cotty
3
.
1
Division of Plant Pathology and Microbiology,
University of Arizona, Tucson, AZ;
2
Center for Food Safety and Applied
Nutrition, Food and Drug Administration, College Park, MD;
3
USDA-ARS,
Division of Plant Pathology and Microbiology, University of Arizona, Tucson,
AZ.
E-3 Infuences of Herbicides on Release of Atoxigenic Strains. Nicholas P.
Garber
1
and Peter J. Cotty
2
.
1
Division of Plant Pathology and Microbiology,
University of Arizona, Tucson, AZ;
2
USDA-ARS, Division of Plant Pathology
and Microbiology, University of Arizona, Tucson, AZ.
E-4 Screening of Atoxigenic Aspergillus favus Isolates for Ability to Inhibit
Afatoxin B
1
Production by Toxigenic Aspergillus favus. A. Jha, R. Sweany,
and K. E. Damann. Department of Plant Pathology and Crop Physiology,
Louisiana State University Agricultural Center, Baton Rouge, LA.
F. Afatoxin - Potential Use of Natural Products for Prevention of Fungal Invasion and/
or Afatoxin Biosynthesis in Crops
F-1 Identifcation of Two Maize Seed Compounds that Infuence Afatoxin
Biosynthesis. Robert A. Holmes
1
, Rebecca S. Boston
1
, and Gary A. Payne
2
.
1
Department of Botany, North Carolina State University, Raleigh, NC;
2
Department of Plant Pathology, North Carolina State University, Raleigh, NC.
F-2 A New Peanut Phytoalexin with Stilbene and Tetronic Acid Moieties. V.
S. Sobolev
1
, S. T. Deyrup
2
, and J. B. Gloer
2
.
1
USDA-ARS, National Peanut
Research Laboratory, Dawson, GA;
2
Department of Chemistry, University of
Iowa, Iowa City, IA.
32
33
G. Afatoxin - Detection, Extraction, and Analysis of Afatoxins
G-1 Examination of Error Components Associated with Quantifcation
of Afatoxin in Ground Corn Grain with In house CD ELISA. M. J.
Clements
1
, G. L. Windham
1
, C. M. Maragos
2
, W. P. Williams
1
, T. D. Brooks
1
, L.
K. Hawkins
1
, and H. M. Gardner
1
.
1
Research Unit, Mississippi State, MS;
2
USDA ARS, National Center for
Agricultural Utilization Research, Peoria, IL.
G-2 Using Hyperspectral Technology to Measure Fungal Growth and Assess
Mycotoxin Contamination of Corn. Z. Hruska
1
, H. Yao
1
, K. DiCrispino
1
, K.
Bradham
1
, D. Lewis
1
, J. Beach
1
, R. L. Brown
2
, and T. E. Cleveland
2
.
1
Institute
for Technology Development, Stennis Space Center, MS;
2
USDA-ARS,
Southern Regional Research Center, New Orleans, LA.
34
:n Axxu~i Fuxc~i Gvxo:ics Vovxsnov 35
Pvocvvuixcs ov :nv :ooAxxu~i Mui:icvov Avi~:oxix/Fu:oxisix ii:ix~:iox a
5
TH
ANNUAL FUNGAL GENOMICS WORKSHOP
Moderator: Roy Cantrcll, Cotton !ncorporatcd
34
Finding Target Genes for Better Control of Aspergillus
Jong H. Kim
!
, 8rucc C. Campbcll
!
, Jiujiang Yu
2
, Grcgory S. May
1
, Kathlccn L. Chan
!
, Gary A.
Paync
4
, ccpak 8hatnagar
2
and Tomas . Clcvcland
2
!
USDA-ARS, Western Regional Research Center, Albany, CA;
2
1
4
Department of
Plant Pathology, North Carolina State University, Raleigh, NC
Gallic acid, lrom hydrolysablc tannins in thc pclliclc ol walnut kcrncls, dramatically inhibits biosynthcsis
ol aatoxin by Aspergillus favus. Tc gcnctic basis lor this inhibition was lound to takc placc upstrcam
lrom thc aatoxin gcnc clustcr, including its rcgulatory gcnc, afR. thcr antioxidant phcnolics showcd
similar antiaatoxigcnic activity as gallic acid. tert8utyl pcroxidc trcatmcnt ol A. favus rcsultcd in an
approximatc doubling ol aatoxin biosynthcsis comparcd to untrcatcd lungi. Tc lact that oxidativc
strcss and antioxidants acct aatoxin biosynthcsis suggcsts that thc antioxidativc rcsponsc systcms
ol A. favus arc targcts lor control. High throughput scrccning, using ycast, Saccharomyces cerevisiae,
as a modcl lungus, quickly idcntincd a numbcr ol lungal gcncs vulncrablc to trcatmcnt by phcnolic
compounds. Tc assay also providcd a mcans to asscss thc bioactivity ol combinations ol phcnolics and
ccrtain lungicidcs accting mitochondrial rcspiration. For cxamplc, thc sod mutant was vcry scnsitivc
to trcatmcnt by ccrtain phcnolics and commcrcial lungicidcs or drugs, strobilurins/antimycin A, both
ol which inhibit complcx !!! ol thc mitochondrial rcspiratory chain. Tis ccctivcncss was vcrincd
by strcssing this systcm in thc targct lungus, A. favus, and using complcmcntation analysis, whcrcin
thc mitochondrial supcroxidc dismutasc (MnS) gcnc (sodA) ol A. favus in thc ortholog mutant,
sod, ol S. cerevisiae, rclicvcd phcnolic induccd strcss. Mitochondrial antioxidativc strcss systcms arc
important in lungal rcsponsc to antilungals. Combincd trcatmcnt ol lungi with phcnolics and inhibitors
ol this rcspiratory systcm ccctivcly supprcsscs growth ol A. favus, syncrgistically. !dcntilying thc gcncs
in othcr antioxidativc rcsponsc systcms in othcr pathogcns should grcatly improvc mcthods lor lungal
control using combinations ol compounds that targct thosc gcncs.
36 :n Axxu~i Fuxc~i Gvxo:ics Vovxsnov
Comparative Genomic Analysis of Secondary Metabolite Gene Clusters of
Closely Related Aspergilli
Villiam C. Nicrman
!
, Natalic . Fcdorova
!
, Cathcrinc M. Ronning
!
, Jcnnilcr Vortman
!
,
Masayuki Mashida
2
, Jiujiang Yu
1
, Tomas . Clcvcland
1
, ccpak 8hatnagar
1
and Gary Paync
4
!
e Institute for Genomic Research, Rockville, MD;
2
Institute for Biological Resources and Functions, National
Institute of Advanced Industrial Science and Technology, Tsukuba, Japan;
1
USDA, ARS, Southern Regional
Research Center, New Orleans, LA;
4
Raleigh, NC
Vc havc pcrlormcd prcliminary comparativc analysis ol thc gcnomc scqucncc ol thrcc closcly rclatcd
Aspcrgilli, Aspergillus fumigatus Alu291, A. fumigatus CA!0, and Neosartorya fscherii NRRL!8!.
Among our obscrvations, wc notcd that spccics and strain spccinc gcncs arc oltcn lound in clustcrs
within polymorphic subtclomcric domains. Somc ol thcsc clustcrs contain sccondary mctabolism
biosynthctic gcncs, which arc onc ol thc most variablc groups ol gcncs in thcsc gcnomcs. Frcqucnt
cxchangcs bctwccn nonhomologous chromosomcs obscrvcd in subtclomcric rcgions may lacilitatc
amplincation and divcrsincation ol sccondary mctabolism and othcr nichc adaptation gcncs in thcsc
lungi. Vith thcsc obscrvations in hand wc arc bcginning to cxplorc thc gcnomcs ol two othcr closcly
rclatcd spccics, A. favus and A. oryzae. Vc havc undcrtakcn to idcntily all ol thc sccondary mctabolitc
clustcrs in both ol thcsc organisms and to comparc thc idcntitics ol thc clustcrs, thcir complctcncss, and
thc syntcnic ordcr ol gcncs within thcsc clustcrs. ur nndings will bc rcportcd and contrastcd to thosc
obscrvcd in thc two A. fumigatus strains and in N. fscheri.
Aspergillus favus Genomics in Discovering Genes Involved in Afatoxin
Biosynthesis
J. Yu
!
, J.R. Vilkinson
!
, V.C. Nicrman
2, 1
, H.S. Kim
2
, G.A. Paync
4
, 8.C. Campbcll
, . 8hatnagar
!
,
and T. . Clcvcland
!
!
USDA/ARS, Southern Regional Research Center, New Orleans, LA;
2
e Institute for Genomic Research,
Rockville, MD;
1
e George Washington University School of Medicine, Department of Biochemistry and
Molecular Biology, Washington DC;
4
USDA/ARS, Western
Regional Research Center, Albany, CA
Aatoxins arc thc most carcinogcnic natural compounds among thc known mycotoxins. Aspergillus
favus produccs aatoxins 8! and 82 and causcs aatoxin contamination ol prcharvcst crops such as
corn, cotton, pcanuts and trccnuts. uc to thc signincant hcalth and cconomic impacts ol aatoxin
contamination, thc chcmistry, cnzymology, and gcnctics ol aatoxin biosynthcsis in A. favus and
A. parasiticus arc bcing activcly studicd. !n A. favus thcrc arc cight chromosomcs with an cstimatcd
gcnomc sizc ol about 1116 Mbp that harbor approximatcly !2,000 lunctional gcncs. Gcnctic studics on
aatoxin biosynthcsis in A. favus and A. parasiticus lcd to thc cloning ol 2 clustcrcd gcncs within a 70kb
NA rcgion rcsponsiblc lor thc cnzymatic convcrsions in thc aatoxin pathway. !dcntincation and
clucidation ol gcncs involvcd in aatoxin biosynthcsis through gcnomics is thc kcy stratcgy in ordcr to
bcttcr undcrstand thc molccular mcchanisms that control or rcgulatc aatoxin production. Tc widcly
uscd wild typc strain A. favus NRRL 117 (ATCC# 20026) was uscd in thc ST projcct. Scqucncing
and annotation ol A. favus STs lrom a normalizcd A. favus cNA library idcntincd 7,2!8 uniquc
ST scqucnccs. Gcncs that arc putativcly involvcd in aatoxin biosynthcsis, rcgulation and signal
transduction, lungal virulcncc or pathogcnicity, strcss rcsponsc or antioxidation, and lungal dcvclopmcnt
wcrc idcntincd lrom thcsc STs. Microarrays containing ovcr ,000 uniquc A. favus gcnc amplicons
wcrc constructcd at Tc !nstitutc lor Gcnomic Rcscarch (T!GR). For gcnc pronling cxpcrimcnts using
microarrays, thc lour basic culturc mcdia wcrc uscd: ycast cxtract (Y, nonaatoxinproducing), ycast
cxtract sucrosc (YS, aatoxinproducing), pcptonc minimal salt (PMS, nonaatoxinproducing), and
glucosc minimal salts (GMS, aatoxinproducing). Tc mycclium was harvcstcd at 48 and 96 hours
rcspcctivcly altcr inoculation. Gcnc pronling undcr aatoxinproducing and nonproducing conditions
has thus lar idcntincd hundrcds ol gcncs that arc potcntially involvcd in aatoxin production. Tc
1NA Array 900MPXTM protocol ol Gcnisphcrc gavc thc bcst rcsults on dctcction scnsitivity. Tcrc
arc 7802 scorablc spots dctcctcd including thc aatoxin pathway gcncs (ovcr 90 ol thc total spots on
thc array). Among thcm, 414 wcrc at signincant lcvcls and 28 STs wcrc lound to bc uprcgulatcd.
Furthcr invcstigations on thc lunctions ol thcsc gcncs by gcnc knockout cxpcrimcnts arc undcrway.
Tis rcscarch is cxpcctcd to providc inlormation lor dcvcloping ncw stratcgics lor control ol aatoxin
contamination ol agricultural commoditics.
Mining Expressed Sequence Tags (ESTs) Leads to Identifcation of Putative
FUM Cluster Transcription Factor
arcn V. 8rown, Robcrt A.. 8utchko, Mark 8usman and Robcrt H. Proctor
Mycotoxin Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL
Fumonisins arc a lamily ol mycotoxins produccd by Fusarium verticillioides (tclcomorph Gibberella
moniliformis) and can bc lound contaminating maizc throughout thc world. F. verticillioides gcncrally
grows in maizc tissuc without causing discasc symptoms, but undcr somc conditions, can causc sccdling
blight and car, root and stalk rots ol maizc as wcll as synthcsizc lumonisins. Fumonisins causc scvcral
dicrcnt toxicoscs in animals and, in humans, arc cpidcmiologically associatcd with csophagcal canccr
and birth dclccts in somc rcgions ol thc world. Fumonisins arc synthcsizcd via cnzymcs cncodcd by
gcncs localizcd within a 42. kb portion ol thc F. verticillioides gcnomc. Undcrstanding lumonisin
biosynthcsis and its gcnctic rcgulation may lcad to thc dcvclopmcnt ol novcl mcthods to prcvcnt
contamination ol maizc with lumonisins and thcrcby climinatc thc toxins lrom thc animal and human
lood chain.
vcr lour ycars ago, wc initiatcd a projcct with Tc !nstitutc lor Gcnomic Rcscarch (T!GR) to
scqucncc thousands ol F. verticillioides cNAs in ordcr to gcncratc an cxprcsscd scqucncc tag (STs)
databasc. Tc primary goal ol this projcct was to idcntily gcncs that rcgulatc lumonisin biosynthcsis. Vc
gcncratcd cight cNA librarics, cach lrom a dicrcnt culturc condition, lrom which T!GR scqucnccd
ovcr !!!,000 cloncs. Altcr analysis, ovcr 87,000 high quality scqucnccs (c.g. STs) wcrc gcncratcd that
corrcspond to !!,!!9 uniquc scqucnccs. Transcripts corrcsponding to gcncs within thc lumonisin gcnc
clustcr wcrc lound primarily in lour ol thc cight librarics. Trcc ol thcsc librarics wcrc constructcd
lrom RNA harvcstcd lrom F. verticillioides grown in GYAM, a liquid mcdium that supports lumonisin
production, lor 24, 48/72, or 96 hours. Tc lourth library was constructcd lrom growth on maizc mcal,
which also supports lumonisin biosynthcsis. Vc rcasoncd that a comparison ol thc STs lrom dicrcnt
librarics would idcntily dicrcntially cxprcsscd gcncs important to lumonisin biosynthcsis. Prcliminary
analysis has lound a numbcr ol candidatc lumonisin rcgulatory gcncs and lunctional analysis is in
progrcss.
Tis rcport dcscribcs analysis ol a sct ol STs that match gcnomic scqucncc adjaccnt to thc lumonisin
gcnc clustcr. 8LAST analysis ol thc gcnomic scqucncc corrcsponding to thc STs indicatcd that it did
not sharc similarity with any prcviously charactcrizcd scqucncc in Gcnc8ank. !n contrast, 8LAST
analysis ol thc STs indicatcd that thcy sharc signincant similarity with transcriptional lactors ol thc
Zn(!!)Cys lamily. Analysis ol thc gcnc structurc idcntincd cight introns ol which two arc locatcd
within thc scqucncc cncoding thc Zn(!!)Cys motil. A majority ol thc STs arc altcrnativc splicc
lorms (ASFs) whcrc an intron was cithcr not cxciscd or utilizcd an altcrnativc 1 splicc scqucncc. All ol
thc ASFs arc unablc to cncodc thc prcdictcd lull lcngth FUM protcin. Tc distribution ol ASFs in
thc GYAM librarics is consistcnt with thc pattcrn ol ASF STs ol lour lumonisin biosynthctic gcncs
and lurthcr support our hypothcsis that ASFs scrvc a biological lunction. Gcnc dclction studics indicatc
that FUM plays an important but not absolutc rolc in lumonisin biosynthcsis as dclction ol FUM
rcduccd lumonisin production to 10 ol wildtypc. Studics arc in progrcss to cxaminc il thc FUM
ASFs scrvc a biological lunction.
Release of the Aspergillus favus Genome Sequence
Gary A. Paync
!
, 8. Pritchard
!
, Jiujiang Yu
2
, Villiam C. Nicrman
1
, Ralph can
!
, ccpak
8hatnagar
2
, and Tomas . Clcvcland
2
!
2
USDA-ARS, Southern
Regional Research Center, New Orleans, LA;
1
e Institute for Genomic Research, Rockville, MD
Aspergillus favus is a pathogcn ol maizc, pcanuts, cottonsccd, and trcc nuts, and contaminatcs thcm
with thc carcinogcn, aatoxin. Tcrc arc no ccctivc control proccdurcs lor thc lungus. To gain a grcatcr
undcrstanding ol thc lactors rcsponsiblc lor pathogcnicity and aatoxin production, a wholc gcnomc
scqucncing projcct lor Aspergillus favus was initiatcd in 2001. Tis projcct, which is dircctcd by Gary
Paync, is lundcd primarily lrom thc USA/NR! Compctitivc Grants Program (USACSRS
Award Numbcr: USA20010428) with supplcmcntal lunding lrom USA/ARS/SRRC in Ncw
rlcans. Tc scqucncing projcct is ncar complction and wc arc now in thc manual annotation and
comparativc gcnomics phasc.
Scqucncing to X covcragc was donc at Tc !nstitutc ol Gcnomic Rcscarch (T!GR) undcr thc
supcrvision ol r. Villiam Nicrman. A multiplc library stratcgy with dicrcnt inscrt sizcs was uscd
to attain maximal gcnomc covcragc and maximal linkagc ol thc asscmblcd contigs. A combination ol
14 kb and !0 kb inscrt sizc librarics and a 0 kb linking library wcrc uscd. Tc scqucncc rcads can bc
obtaincd lrom NC8!.
Automatcd annotation was donc undcr thc supcrvision ol r. Jcnnilcr Vortman at T!GR using
annotation tools traincd on gcnomic scqucncc ol A. oryzae as wcll as A. favus and A. oryzae STs. r.
Jiujiang Yu at thc USA/ARS/SRRC dircctcd thc scqucncing ol thc A. favus STs, which havc bccn
critical to gcnc annotation. Finc nnishing ol thc scqucncc, which includcs closing thc small gaps is ncar
complction.
A wcb browscr was dcvclopcd at North Carolina Statc Univcrsity undcr thc dircction ol 8cth
Pritchard that allows 8last matchcs to gcncs, protcins and gcnomic scqucncc ol othcr Aspergillus spccics,
alignmcnts ol STs, and G annotations. Ncw annotations will bc updatcd on thc sitc. Links to thc wcb
browscr and to othcr inlormation on thc scqucncing projcct can bc lound at: www.aspcrgillusavus.org.
Tc gcnomc has bccn asscmblcd into 79 scaolds ranging in sizc lrom 4. Mb to !.0 Kb. vcr
7 ol thc gcnomc is rcprcscntcd in thc !0 largcst scaolds. Tc cstimatcd gcnomc sizc ol 16.1 Mb is
similar to that lor A. oryzae (16.8 Mb), a closcly rclatcd spccics. Tcsc two lungi arc cnrichcd in gcncs
lor sccondary mctabolism. A. favus, lor cxamplc, is prcdictcd to havc 14 polykctidc synthascs, 22 non
ribosomc pcptidc synthascs, 77 A8C transportcrs and !22 cytochromc p40 cnzymcs.
Aspergillus favus and A. oryzae (thc prcdominatc lungus uscd in lood lcrmcntation) arc closcly rclatcd
and arc likcly ccotypcs. A comparison ol thc gcnomcs ol thcsc two lungi will likcly rcvcal inlormation
on changcs that havc occurrcd during thc domcstication ol A. oryzae, and hclp idcntily pathogcnicity
lactors in A. favus.
Production of Cyclopiazonic Acid, Afatrem and Afatoxin is Regulated by veA,
a Gene Necessary for Sclerotial Formation in Aspergillus favus
R.M. uran
!
, J.V. Cary
2
, and A.M. Calvo
!
!
Department of Biological Sciences, Northern Illinois University. DeKalb, IL;
2
Food and Feed Safety Research
Unit, USDA, ARS, Southern Regional Research Center. New Orleans, LA
Targcting gcncs involvcd in Aspcrgillus mycotoxin biosynthcsis and/or lungal dcvclopmcnt would
contributc to rcducing thc dctrimcntal cccts ol thcsc mycotoxins. Contamination ol crops such
as cotton, corn, sorghum, pcanuts, walnuts and othcr oil sccds by aatoxin and othcr Aspcrgillus
mycotoxins lcads to signincant cconomic losscs in thc U.S. and a hcalth thrcat in dcvcloping countrics.
!t is wcll cstablishcd that thc biosynthcsis ol natural products, including mycotoxins, is associatcd with
lungal dcvclopmcnt. Vc havc lound that thc Aspergillus favus veA gcnc rcgulatcs both, lormation ol
rcsistant structurcs (sclctoria), and mycotoxin biosynthcsis. ur rcsults dcmonstratcd that thc A. favus
veA mutant is unablc to producc aatoxin, whilc aatoxin 8, 8, G and G wcrc dctcctcd in thc veA
wild typc strain. Tc cxprcssion ol afR (cncoding lor a spccinc transcription lactor that activatcs thc
aatoxin gcnc clustcr) and aatoxin cnzymatic gcncs was blockcd in thc veA mutant.
ur chcmical analysis also indicatcd thc abscncc ol othcr mctabolitcs in thc veA mutant. For this
rcason wc also cxamincd whcthcr veA is involvcd in rcgulating thc production ol othcr toxins in A.
favus. Spccincally, wc cvaluatcd thc production ol cyclopiazonic acid and aatrcm. Cyclopiazonic acid
is a spccinc inhibitor ol calciumdcpcndcnt

ATPasc and induccs altcrations in ion transport across ccll

mcmbrancs. !ntcrcstingly, our rcsults showcd that thc A. favus veA mutant prcscnts a dccrcasc in CPA
production with rcspcct to thc wild typc strain. !n thc casc ol aatrcm, a potcnt trcmorgcnic mycotoxin,
its production was complctcly blockcd in thc veA mutant, indicating that veA is also rcquircd lor
thc synthcsis ol this mycotoxin. Tc aatrcm gcnc clustcr (containing atm gcncs) has bccn rcccntly
idcntincd and charactcrizcd. Vc lound that thc atm gcncs arc not cxprcsscd in thc A. favus veA mutant.
Tc lact that veA not only rcgulatcs aatoxin production but also othcr mycotoxins adds to thc valuc ol
veA as a possiblc control targct. veA involvcmcnt in thc production ol A. favus mycotoxins is consistant
with our prcvious obscrvations: dclction ol veA in Aspergillus parasiticus and Aspergillus nidulans veA
mutants rcsults in climination ol aatoxin and stcrigmatocystin production rcspcctivcly.
Furthcrmorc, targcting veA could also aid in dccrcasing A. favus survivability by accting sclcrotial
dcvclopmcnt. Sclcrotia arc rcsistant structurcs that allow thc lungus to survivc undcr advcrsc conditions
until thc cnvironmcnt is lavorablc again lor growth and plant inlcction. Molccular studics ovcr thc
control ol sclcrotial dcvclopmcnt arc limitcd. Vc havc shown that thc A. favus veA mutant, as with
A. parasiticus, is complctcly unablc to producc sclcrotia, indicating that thc veA gcnc is csscntial lor
sclcrotial morphogcncsis.
Tc impact ol this study on lungal discasc rcsistancc may wcll go bcyond control ol aatoxin
contamination ol lood and lccd crops. Rcccntly, wc havc lound veA homologucs across lungal gcncra,
including spccics ol agricultural and mcdical importancc. 8ccausc veA has only bccn lound in lungi,
thcrc is a grcat potcntial lor using veA as a targct lor plant, animal and human discasc control. Futurc
studics might includc thc gcncration ol transgcnic plants rcsistant to lungal invasion and toxin
production that arc dcvclopcd bascd on thc production ol inhibitors ol veA. Additionally, this rcscarch
might lcad to anti\cA lungal drugs that could bc uscd to prcvcnt human and animal discasc.
PANEL DISCUSSION: Fungal Genomics Workshop
Panel Chair: Gary Paync, North Carolina Statc Univcrsity
Panel Members: 8rucc Campbcll, Villiam Nicrman, Jiujiang Yu, arcn 8rown & Ana Calvo
Questions for William Nierman
Q: Arc thcrc common tclomcrc scqucncc rcpcats:
A: Ycs, and wc usc that lor gcnotyping thcsc rcgions. Gcnc rcarrangcmcnts mcdiatcd by thcsc rcpcats
may drivc cnrichmcnt ol uniquc clustcrs ol gcncs in thcsc rcgions. l coursc, this is my spcculation.
Q: !s it possiblc to comparc tcrtiary scqucncc that is not usablc at thc nuclcotidc or amino acid lcvcl:
A: Tcrc has bccn a lot ol discussion about this but probably not cnough structurcs prcscnt to run thc
tcchnology yct.
Questions for Bruce Campbell
Q: ocs thc addition ol antioxidants acct lungal growth:
A: Not at lcvcls wc arc tcsting.
Q: Vill incrcasing antioxidant lcvcls incrcasc rcsistancc in plants:
A: Ycs
Q: o you think that aatoxin plays a rolc in oxidativc strcss:
A: Ycs
Q: Havc you comparcd thc rcsponsc ol aatoxin nonproducing mutants with wild typc strains lor thcir
rcsponsc to oxidativc strcss:
A: No. Tcrc may bc othcr ways thc atoxigcnic strains rcspond to oxidativc strcss othcr than aatoxin
biosynthcsis. !m surc cvcn thc toxigcnic strains havc othcr mcchanisms, as wcll.
Questions for Jiujiang Yu
Q: Can you gain morc inlormation lrom thc Aymctrix oligo arrays or lrom amplicon arrays:
A: Tcrc is a widcr rangc ol intcnsitics and it is casicr to spot and optimizc amplicon arrays.
Commcnt lrom Nicrman: Fungal arrays prcscnt ncw problcms in that somc oligos may not match
mcssagc bccausc wc havc a hardcr timc with gcnc annotation ol splicc boundarics and maturc mcssagc.
Vc still lack somc powcr on gcnc calling. icrcnt gcnc calling programs givc dicrcnt , 1 boundarics
and splicc sitcs.
Questions for Ana Calvo
Q: o you havc thc scqucncc lor vcA:
A: Ycs
Q: Vhat is thc rclationship bctwccn clcistothccia production and mycotoxin biosynthcsis:
A: Morphogcncsis is oltcn associatcd with mycotoxin production, c.g. \cA is ncccssary lor both
mycotoxin production and clcistothccial production. Tcrclorc, wc cxpcct gcnctic links. For cxamplc,
thc FadA pathway links ascxual structurcs and mycotoxins. Tc link is not wcll dcnncd lor scxual
structurcs at this timc. Additionally, sclcrotial dcvclopmcnt is not wcll charactcrizcd in gcncral.
Q: Sclcrotia production is linkcd with aatoxin production but many strains lack sclcrotia and still
producc aatoxin. ocs thc veA dclction strain producc aatoxin: ocs thc mutant losc thc ability to
producc conidia:
A: Scvcral proccsscs arc ncccssary lor sclcrotial production and veA contributcs to thcsc proccsscs. Tc
veA dclction mutant docs producc conidia.
Q: Vhat is thc rolc ol light in your lungus: !s it rclatcd to strcss:
A: ur hypothcsis is that in A. nidulans light is a signal to dircct thc lormation ol acrial sporcs il thc
lungus is ncar thc surlacc and survival structurcs il thc lungus is dccpcr in thc soil. Scvcral photorcccptors
havc bccn idcntincd but wc arc not surc which oncs acct vcA.
Q: o you think that mycotoxins arc rclatcd to survival ol thcsc structurcs sincc thcir production is
induccd at thc soil surlacc:
A: Maybc
Commcnt lrom Campbcll: Vc havc lound that oxidativc strcss incrcascs sclcrotial dcvclopmcnt and
mclanization. Antioxidants supprcss not only aatoxin biosynthcsis, but also mclanization and sclcrotial
dcvclopmcnt suggcsting that sclcrotial dcvclopmcnt and aatoxin biosynthcsis might havc somc
common triggcring mcchanism.
Questions for Daren Brown
Q: !s Fum 2! in othcr lungi:
A: Ycs, thcrc arc somc lumonisinlikc clustcrs in othcr lungi.
Evolutionary Processes in the Afatoxin Gene Cluster in Aspergillus
!. Carbonc
!
, J.L. Jakobck
!
, .H. Moussa
!,2
, J.. Cox
!
and 8.V. Horn
1
!
Center for Integrated Fungal Research, Department of Plant Pathology, North Carolina State University,
Raleigh, NC;
2
Present address: Faculty of Sciences II, Department of Biological Sciences, Lebanese University,
Beirut, Lebanon;
1
National Peanut Research Laboratory, USDA, ARS, Dawson, GA
Aatoxins arc potcnt natural carcinogcnic sccondary mctabolitcs produccd by scvcral spccics in thc
gcnus Aspergillus. Rcccntly nonaatoxigcnic A. favus strains wcrc approvcd as biocontrol agcnts lor
usc on cotton and pcanuts. Although thcsc biocontrol strains havc provcn to bc ccctivc, wc havc no
knowlcdgc ol thc longtcrm cccts that introduccd strains havc on thc cvolution ol aatoxigcnicity. ur
hypothcsis is that a low lcvcl ol rccombination and gcnc ow among Aspergillus spccics is signincantly
contributing to thc pcrsistcncc and lurthcr cvolution ol aatoxigcnic strains. Vc arc currcntly tcsting
this hypothcsis by locusing on A. favus and A. parasiticus, thc two most abundant aatoxigcnic spccics.
ur cxamination ol nuclcotidc scqucncc variation in 2! intcrgcnic rcgions across thc cntirc aatoxin
gcnc clustcr ol A. parasiticus indicatcs thc prcscncc ol rccombination blocks and thcrc is cvidcncc that
balancing sclcction has inucnccd gcnctic variation in thcsc blocks. Tc samc blocks appcar to bc
conscrvcd lor putativc orthologs ol thcsc gcncs in A. nidulans, A. favus, and A. fumigatus, indicating thc
potcntial lor an introduccd biocontrol strain to acquirc toxigcnicity gcncs lrom indigcnous strains via
rccombination or lrom sympatric spccics via horizontal translcr. Vc arc currcntly cxamining thc timing
and lrcqucncy ol thcsc cvcnts in naturc.
Diferential Gene Expression Levels for Aspergillus favus Resistance in Two
Inbred Maize Lines
R. Y. Kcllcy
!
, . L. 8oykin
2
, L. K. Hawkins
1
and V. P. Villiams
1
!
Dept of Plant and Soil Sciences, Mississippi State University, Mississippi State, MS;
2
USDA-ARS Mid
South Area Statistics Ofce, Stoneville, MS;
1
USDA-ARS Corn Host Plant Resistance Research Unit,
Mississippi State, MS
cNA microarray is a powcrlul gcnc cxprcssion tool which allows invcstigators to cxaminc changcs in
thc cxprcssion lcvcls ol thousands ol gcncs simultancously. cNA microarrays and othcr gcnomicscalc
hybridizationbascd tcchnologics havc improvcd throughput, scnsitivity, and vcrsatility lor idcntilying
dicrcntially cxprcsscd gcncs. For cxamplc, microarray analysis has bccn uscd lor pronling RNA lcvcls
in Arabidopsis, bactcria, ycast, and mammals. !n maizc, microarray tcchnology has bccn applicd to
cxaminc gcnc cxprcssion in rcsponsc to discasc, growth, watcrdcncicnt conditions and strcss scnsitivity.
Rcccntly, gcncs dicrcntially cxprcsscd in Aspergillus favus during aatoxin biosynthcsis wcrc idcntincd
using microarray, but no work has bccn donc using microarray to look at gcnc cxprcssion rclatcd to A.
favus inlcction and aatoxin production in maizc. Tcrclorc, thc objcctivc ol this cxpcrimcnt was to
idcntily dicrcntially cxprcsscd gcncs lor A. favus rcsistancc in thc \a1 (susccptiblc) and Mp1!1
(rcsistant) maizc (Zea mays L.) lincs using cNA microarray analysis. Primary cars lrom plants in
trcatcd plots wcrc inoculatcd with isolatc NRRL 117 ol A. favus !4 days altcr pollination and wcrc
harvcstcd two days altcr inoculation. Uninoculatcd cars wcrc harvcstcd !6 days altcr pollination and
uscd as a control. cNA lrom thc inoculatcd and uninoculatcd cars was labclcd with Cy1 and Cy
uorcsccnt dycs. 8oth samplcs wcrc hybridizcd to thc unigcnc !.0!.0 maizc chip containing ,06 ST
contigs lrom ST librarics dcrivcd lrom immaturc lcal, cndospcrm, immaturc car and thc root.
ut ol thc 06 STs analyzcd, !21 or 2.4 ol thc total STs analyzcd wcrc signincantly uprcgulatcd
lor thc susccptiblc inbrcd linc \a1 and 9 or !.8 ol thc total STs analyzcd wcrc signincantly up
rcgulatcd lor thc rcsistant inbrcd linc Mp1!1, and !6 or 0.1 ol thc total STs wcrc uprcgulatcd
lor both thc susccptiblc and rcsistant inbrcd lincs. Tc cxprcsscd STs includcd gcncs with known
lunctions such as strcss rcsponsc, mctabolism, protcin synthcsis, ccllular communication and signal
transduction, transcription and RNA proccssing and photosynthcsisassociatcd gcncs. Uprcgulatcd
STs also includcd gcncs with unknown lunctions, dcmonstrating thc usclulncss ol microarray as a
gcnc discovcry tool.
Tis study providcs charactcrization ol thc maizc car cxprcssion pattcrns lor a numbcr ol maizc gcncs
whcn cxposcd to A. favus. For a substantial numbcr ol thc gcncs studicd, prcviously publishcd data arc
availablc on thcir cxprcssion pattcrns in othcr tissucs but not whcn cxposcd to A. favus, thcrclorc, thc
currcnt data providc initial inlormation usclul toward thc charactcrization ol gcncs cxprcsscd in maizc
cxposcd to A. favus at two days altcr inoculation.
Enhanced Activity of Fungicides by Positive Interaction with Natural Phenolic
Agents: Target-gene Based Bioassays for Control of Aspergilli
Jong H. Kim
!
, 8rucc C. Campbcll
!
, Jiujiang Yu
2
, Norccn Mahoncy
!
, Kathlccn L. Chan
!
, Russcll
J. Molyncux
!
, ccpak 8hatnagar
2
, Tomas . Clcvcland
2
, Grcgory S. May
1
and Gary A.
Paync
4
!
USDA-ARS, Western Regional Research Center, Albany, CA;
2
1
4
Department of
Plant Pathology, North Carolina State University, Raleigh, NC
Signal transduction and strcssrcsponsc gcncs ol lungal pathogcns play important rolcs lor cxcrting
pathogcncsis and, in somc cascs, biosynthcsis ol mycotoxins. As such, thcy may scrvc as potcntially
viablc targcts lor antilungal compounds. Rcsults ol our rcscarch, as prcscntcd in this postcr, show that
targcting gcncs in thc mitochondrial rcspiratory chain pathways, MAPK or vacuolar H()ATPasc (\
ATPasc) using salc, natural compounds can signincantly clcvatc thc scnsitivity ol lungi to commcrcial
lungicidcs or drugs. Tc usc ol such compounds can rcsult in lowcring ccctivc dosagcs, costs ol
trcatmcnt and potcntial lor dcvclopmcnt ol rcsistancc.
Ccllular targcts ol scvcral convcntional lungicidcs arc alrcady known. xamplcs includc macro
molccular synthcsis (e.g., nuclcic acids, amino acids, ccll wall, etc.), ccll division, signal transduction
and rcspiration. clccts in any ol thcsc systcms can lcad to oxidativc strcss, with a rcsultant dccrcasc
in ccll viability. Many dclcnsivc phcnolic compounds arc produccd or rclcascd by plants during lungal
inlcction. Vc thcorizc that disruption ol ccllular rcdox homcostasis using phcnolics may inhibit lungal
growth by disruption ol ccllular rcdox homcostasis.
Tc molccular targct lor strobilurinrclatcd lungicidcs, such as azoxystrobin or krcsoximmcthyl, is thc
mitochondrial rcspiratory bc complcx. !nhibition ol this complcx cvcntually lcads to ccllular oxidativc
strcss causcd by abnormal rclcasc ol clcctrons lrom thc rcspiratory chain. Using dclction mutants, wc
lound at lcast nvc phcnolic compounds that disrupt thc normal lunction ol mitochondrial rcspiration
ol ycast. Combincd trcatmcnts ol thcsc phcnolic agcnts and commcrcially availablc lungicidcs that arc
inhibitors ol thc mitochondrial rcspiratory chain havc a !00 to !000lold syncrgistic lungicidal ccct
duc to disruption ol rcspiration and inhibiting thc oxidativc strcssrcsponsc ol thc lungus. Vc also lound
that thc sakA (MAPK mutant) strain ol A. fumigatus was much morc scnsitivc to phcnolics, indicating
sakA likcly is involvcd in rcgulation ol thc antioxidativc strcss rcsponsc systcm in A. fumigatus, pcrhaps
involving mitochondrial lunction/rcspiration. !n addition, wc lound that thc alkaloid bcrbcrinc targcts
thc activity ol oxidativc strcss gcncs, and combincd trcatmcnt ol this alkaloid and ccrtain phcnolics
rcsultcd in > !0,000 timcs grcatcr lungicidal activity than cithcr compound alonc.
Vc concludc that natural compounds (i.e., phcnolics or alkaloids) can bc dcvclopcd as usclul anti
lungal agcnts with thc molccular targct idcntincd, lcading to ccctivc control ol a broad spcctrum ol
lungal pathogcns.
Deletion of GBP1, a Gene Encoding a Monomeric G Protein, De-represses
Fumonisin Biosynthesis in Fusarium verticillioides
U.S. Sagaram and V.8. Shim
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX
Fumonisins arc a group ol mycotoxins produccd by Fusarium verticillioides (pcrlcct stagc Gibberella
moniliformis) that contaminatc maizc and othcr ccrcal crops. Among thc 8scrics lumonisins, which
occur undcr natural and laboratory conditions, lumonisin 8 (F8) is thc most abundantly produccd
and most toxic lumonisin. Tc occurrcncc ol F8 has bccn linkcd to a numbcr ol human and animal
disordcrs worldwidc. To datc, scicntists havc idcntincd thc lumonisin biosynthcsis gcnc clustcr that is
rcsponsiblc lor synthcsizing lumonisins in F. verticillioides. Howcvcr, wc still lack dctailcd undcrstanding
ol how thc lungus rccognizcs thc cxtcrnal cnvironmcntal/host signals to rcgulatc F8 biosynthcsis. !n
this study, wc dcscribc thc molccular charactcrization ol G8P! and its rolc in F8 biosynthcsis and
conidiation in F. verticillioides. GBP was idcntincd as an cxprcsscd scqucncc tag (ST) uprcgulatcd in
F. verticillioides fcc mutant that showcd rcduccd conidiation and no F8 biosynthcsis. Tc objcctivc ol
this study was to tcst whcthcr GBP is involvcd in F8 biosynthcsis or conidiation, or both. Scqucncc
analysis showcd that GBP is a monomcric Gprotcin that cncodcs a putativc 168amino acid protcin
with similarity to RG and bg subclasscs ol Gprotcins that arc involvcd in dcvclopmcnt and strcss
rcsponscs. clction mutant ol GBP (gbp) cxhibitcd normal growth whcn grown on sclcct dcnncd
mcdia and corn kcrncls. gbp produccd 8 grcatcr F8 lcvcl than thc wild typc whcn grown on corn
kcrncls. Complcmcntation ol gbp with wildtypc GBP rcstorcd F8 production to that ol wild
typc. Tc data indicatcs that dclction ol GBP rcsults in incrcascd F8 production but docs not acct
conidiation. Tc dclction ol GBP did not acct thc cxprcssion lcvcl ol kcy F8 biosynthctic gcncs
(FUM, FUM, and FUM) and ZFR, a positivc rcgulator ol lumonisin biosynthcsis, suggcsting that
thc incrcascd F8 production in gbp is modulatcd via FUM gcnc clustcrindcpcndcnt mcchanism.
Furthcr studics arc ncccssary to clcarly dcnnc thc rolc ol GBP in F8 rcgulation.
A Link between Rho-Signaling and Afatoxin Biosynthesis in Aspergillus favus
. Ryan Gcorgianna
!,2
, Michacl S. Pricc
!,1
, and Gary A. Paync
!,2
!
North Carolina State University, Center for Integrated Fungal Research and Department of Plant Pathology,
Raleigh, NC;
2
North Carolina State University, Genomic Sciences Graduate Program, Raleigh, NC;
1
Duke
University Medical Center, Department of Molecular Genetics and Microbiology, Durham, NC
Tc transition bctwccn growth, dcvclopmcnt, and sccondary mctabolism in nlamcntous lungi is
mcdiatcd by complcx signaling pathways. Rcccntly, wc idcntincd a componcnt ol a signaling pathway
that appcars to modulatc aatoxin production. Transcriptional analysis ol Aspergillus favus grown on
mcdia conducivc or nonconducivc lor aatoxin biosynthcsis rcvcalcd a gcnc whosc cxprcssion parallclcd
that ol thc aatoxin pathway rcgulatory gcnc afR. Tis idcntincd gcnc had thc grcatcst similarity to
rdi, a ycast gcnc that cncodcs a Rhoguanidinc nuclcotidc dissociation inhibitor (RhoG!). clction
ol this gcnc (dcsignatcd afrdiA) in A. favus rcsultcd in a 97 rcduction in aatoxin production. Tc
dclction also causcd a scvcrc growth dclcct on minimal mcdia, a modcratc dclcct on complctc mcdia,
and a tcmpcraturc scnsitivc phcnotypc. Tcsc obscrvcd growth phcnotypcs in thc A. favus rdiA strain
wcrc not similar to thc rcportcd phcnotypc lor thc Saccharomyces cerevisiae rdi null mutant. A similar
mutant phcnotypc was idcntincd in ycast rcsulting lrom dclction ol a gcnc cncoding a RhoGTPasc
associatcd protcin, 8cm4p. A blast scarch ol thc A. favus gcnomc rcvcalcd no scqucnccs with signincant
similarity to bem, including afRdiA. Although bclicvcd not to bc a RhoG!, thc ycast 8cm4p is
thought to havc intcractions similar to thosc ol thc ycast Rdi!p, both known to bc capablc ol associating
with guanidinc nuclcotidc bound Cdc42p and Rho!p. Vc proposc a modcl in A. favus using inlcrcnccs
lrom thc ycast Rhosignaling pathway and ycast systcmic dclction projcct whcrc alRdiA plays a ccntral
rolc in combining known intcractions ol thc A. favus protcins AR and RasA with homologs ol thc
ycast protcins Rho!, Cdc42, and 8bc! lor lacilitating signaling control ol aatoxin production through
a Rhomcdiatcd pathway.
e NADH oxidase, NadA, and its Role in Afatoxin Biosynthesis
Carric Jacobus
!
, Gary Paync
2
, Niki Robcrtson
!,1
!
Department of Genetics, North Carolina State University, Raleigh, NC;
2
Department of Plant Pathology,
1
Department of Botany, North Carolina State University,
Raleigh, NC
Tc nadA gcnc is part ol a gcnc clustcr lor sugar mctabolism that lics adjaccnt to thc aatoxin gcnc
clustcr in Aspergillus favus, A. parasiticus, and A. nomius. Tc cnzymc cncodcd by nadA convcrts NAH
to NA
, colactors nccdcd lor ccrtain rcactions in thc aatoxin biochcmical pathway. Microarray
cxpcrimcnts comparing gcnc cxprcssion bctwccn a wild typc strain ol A. parasiticus and an afR dclction
mutant showcd that nadA cxprcssion was signincantly rcduccd in thc mutant background. Tcsc rcsults
wcrc connrmcd by quantitativc RTPCR and arc consistcnt with thc prcscncc ol a putativc AR
binding sitc upstrcam ol thc coding rcgion ol nadA. Vc hypothcsizcd that NadA may bc nccdcd to
supply NA
colactors lor thc aatoxin biosynthctic pathway and hcncc is uprcgulatcd by AR. !n
ordcr to invcstigatc a conncction bctwccn nadA cxprcssion and aatoxin production, a gcnc rcplaccmcnt
construct was uscd to knock out nadA cxprcssion in A. favus. Aatoxin production in thc prcscncc ol
sucrosc, lructosc, or glucosc was invcstigatcd in thrcc indcpcndcnt nadA mutants. !n cach casc, aatoxin
lcvcls in thc mutant strains wcrc similar to thosc produccd by thc wild typc strain. Tis suggcsts that
NAH oxidasc activity is somchow compcnsatcd lor in thc mutants or that thc NAH oxidasc is
not rcquircd lor aatoxin production. !nvcstigations arc currcntly undcrway to charactcrizc additional
phcnotypcs ol thcsc mutants to bcttcr undcrstand thc rolc ol NA
in aatoxin biosynthcsis.
Metabolic Profling of Aspergillus favus during Afatoxin Biosynthesis
Norm Glassbrook and Gary A. Paync
North Carolina State University, Raleigh, NC
Mctabolic pronling tcchniqucs wcrc applicd to charactcrizing Aspergillus favus during undcr growth
conditions conducivc to aatoxin production (28 C) and thosc not conducivc to production (17 C).
Samplcs analyzcd by gas chromatography couplcd with mass spcctromctry (GC/MS) or liquid
chromatography couplcd with mass spcctromctry (LC/MS).
icrcnccs wcrc obscrvcd in thc small molcculc composition ol Aspergillus grown undcr conditions
conducivc to aatoxin production (28 C) and that grown undcr conditions not conducivc to production
(17 C).
bscrvcd changcs in mctabolitc lcvcls wcrc mappcd onto biochcmical pathways along with rcsults ol
microarray analyscs ol comparablc cxpcrimcnts. Tis typc ol pathway mapping providcs a lramcwork
lor sorting largc quantitics ol mctabolic pronling data and lor lormulating tcstablc hypothcscs.
50
6:n Axxu~i Fu:oxisix ii:ix~:iox Vovxsnov 51
6
TH
Moderator: Larry Antilla, Arizona Cotton Rcscarch and Protcction Council
50
Kernel Constituents Induce Fumonisin Production during Colonization by
Fusarium verticillioides
Charlcs Voloshuk and 8urt 8luhm
Purdue University, West Lafayette, IN
Qucstions rcmain unanswcrcd rcgarding thc intcractions bctwccn thc maizc kcrncls and Fusarium
verticillioides that lcad to thc accumulation ol lumonisins. Vc havc cvaluatcd thc rolc ol kcrncl
cndospcrm composition in rcgulating lumonisin 8 (F8) biosynthcsis. Vc lound that kcrncls lacking
starch duc to physiological immaturity did not accumulatc F8. Quantitativc PCR analysis indicatcd
that kcrncl dcvclopmcnt also acctcd thc cxprcssion ol lungal gcncs involvcd in F8 biosynthcsis,
starch mctabolism, and nitrogcn rcgulation. A mutant strain ol F. verticillioides with a disruptcd alpha
amylasc gcnc was impaircd in its ability to producc F8

on starchy kcrncls, and both thc wildtypc and
mutant strains produccd signincantly lcss F8

on a highamylosc kcrncl mutant ol maizc. Vhcn grown
on a dcnncd mcdium with amylosc as thc solc carbon sourcc, thc wildtypc strain produccd only tracc
amounts ol F8, whcrcas it produccd largc amounts ol F8

whcn grown on amylopcctin. Furthcrmorc,
thc addition ol dcxtrin to amylosc induccd F8

production. Vc concludc that cnzymatic hydrolysis ol
amylopcctin induccs F8

production in F. verticillioides.
52 6:n Axxu~i Fu:oxisix ii:ix~:iox Vovxsnov
Genetics and Breeding of Host Resistance to Fusarium Ear Rot and Fumonisin
Contamination
J.8. Holland
USDA-ARS, Plant Science Research Unit, Department of Crop Science, North Carolina State University,
NC
To invcstigatc thc inhcritancc ol rcsistancc to Fusarium car rot and lumonisin contamination in
maizc, wc cstimatcd hcritabilitics and gcnctic corrclations ol thcsc two aspccts ol discasc in two maizc
populations. nc population ol 2! 8C!S! lincs dcrivcd lrom thc nrst backcross ol G440 to FR!064
(GFR population) was dcvclopcd by r. on Vhitc), and was tcstcd in rcplicatcd, doublcinoculatcd
ncld trials in lour cnvironmcnts. Tc sccond population was !41 rccombinant inbrcd lincs dcrivcd lrom
thc cross ol NC100 to 8!04 (NC8 population), dcvclopcd by r. Goodman, and cvaluatcd in thrcc
cnvironmcnts. Hcritabilitics ol car rot and lumonisin contcnt in thc GFR population wcrc 0.7 and
0.47, rcspcctivcly, and thcir gcnctic corrclation was 0.96. !n thc NC8 population, car rot and lumonisin
contcnt hcritabilitics wcrc 0.86 and 0.80, rcspcctivcly, and thcir gcnctic corrclation was 0.87. Tcsc
rcsults suggcst that dircct sclcction lor rcduccd lumonisin contcnt is thcorctically optimal lor rcducing
susccptibility to lumonisin contcnt, but that indircct sclcction against car rot may bc cconomically most
cmcicnt at rcducing lumonisin contcnt bccausc car rot is much casicr and lastcr to scorc than lumonisin
contcnt. 8oth populations wcrc also nngcrprintcd with at lcast !0 SSR markcrs. QTL lor both traits
wcrc mappcd in both populations, and many QTL lor lumonisin wcrc also dctcctcd in thc samc rcgions
as QTL lor car rot. Howcvcr, somc QTL appcarcd to havc cccts on car rot but not lumonisin, and vicc
vcrsa. Tcrclorc, indircct sclcction on car rot may not bc ccctivc at sclccting lor all ol thc lumonisin
rcducing allclcs. QTL had rclativcly small cccts (maximum ol !8 ol phcnotypic variation) and wcrc
largcly dicrcnt bctwccn populations, so markcrassistcd sclcction will bc hindcrcd by thc gcnctic
complcxity ol rcsistancc to both traits.
NIR Spectroscopy as a Tool for Optimizing Sorting of White Corn Kernels
Contaminated with Fumonisin
T.C. Pcarson
!
and .T. Vicklow
2
!
USDA-ARS, Grain Marketing Research and Production Research Center, Manhattan, KS;
2
USDA-ARS,
National Center for Agricultural Utilization Research, Peoria, IL
Ncar inlrarcd and rccctancc spcctra (00!700 nm) wcrc analyzcd to dctcrminc il thcy could bc uscd
to idcntily singlc wholc whitc corn kcrncls contaminatcd with lumonisin. Kcrncls uscd lor thc study
wcrc obtaincd lrom proccssors in !llinois, !ndiana, Kcntucky, and Ncbraska. Kcrncls wcrc visually
cxamincd and groupcd into six symptom catcgorics: asymptomatic, chalky tip cnd, ycllowtan tip cnd,
rcd strcaks, 0 discolorcd, and !00 discolorcd. Friablc kcrncls and lragmcnts wcrc not includcd in
this study as thcy arc usually rcmovcd by cxisting clcaning cquipmcnt at grain clcvators. Spcctra wcrc
acquircd on both thc gcrm sidc and cndospcrms sidc ol cach kcrncl. Altcr spcctra acquisition, kcrncls
wcrc wcighcd individually and thcn placcd in groups ol nvc according to thcir classincation bascd upon
symptoms ol lungal inlcction and numcrical scqucncc within cach pill box. Total lumonisin (8, 8,
and 8) was mcasurcd with a uoromctcr altcr cxtracts wcrc purincd with immunoamnity columns
(Fumonitcst, \icam, Vatcrtown, MA) using thc proccdurc rccommcndcd lor corn, sorghum, and !7
protcin poultry lccd. Tc lumonisin lcvcl ol cach nvckcrncl group thcn was assigncd to cach individual
kcrncl lrom that group. Kcrncls wcrc analyzcd in groups instcad ol individually to rcducc cost and
analysis timc. Mycological cvaluations, pcrlormcd on grain subsamplcd lrom cach symptom catcgory
and statc, rcvcalcd that thc nvc kcrncl groupings risk producing lalsc positivcs.
For high spccd sorting opcrations, wholc spcctra cannot bc acquircd at throughput ratcs that
arc cconomically lcasiblc. Most commcrcial sorting machincs arc ablc to only mcasurc onc spcctral
band ol light whilc somc machincs can mcasurc two bands. iscriminatc analysis was uscd to sclcct
thc optimal pair ol wavclcngths to idcntily kcrncls containing lumonisin. !t was lound that using
thc wavclcngth pair ol 00nm and !200nm, approximatcly 77 ol thc kcrncls having high lcvcls
ol lumonisin (> 40ppm) wcrc corrcctly classincd. Additionally, approximatcly 96 ol thc kcrncls
having low lcvcls ol lumonisin (< 2 ppm) wcrc corrcctly classincd. !n contrast, il only a singlc band is
sclcctcd lor distinguishing contaminatcd kcrncls, thc accuracy lor kcrncls having low lumonisin lcvcls
(< 2 ppm) drops to approximatcly 81. Tus, usc ol a dual band sorting machinc lor rcmoval ol whitc
corn contaminatcd with lumonisin would rcsult in !1 lcss good product bcing rcmovcd than with a
monochromatic sortcr.
Prcvious work with ycllow corn showcd that approximatcly 8 ol thc aatoxin and lumonisin could
bc rcmovcd by high spccd sortcrs using thc spcctral bands ol 70nm and !200nm. !t was hypothcsizcd
that thc 70nm band was dctccting somc color changcs in lungal inlcstcd kcrncls whilc thc !200nm
band was rcsponding to incrcascd porosity ol thc dcgradcd cndospcrm. !nscct damagcd kcrncls havc
low absorbancc at !200nm, duc to lccding and lungal inlcstation, and would all bc rcjcctcd. !n thc casc
ol whitc corn, 00 nm was lound to bc morc accuratc than 70nm lor thc visiblc spcctral band. Tis may
bc duc to thc whitc corn gcrm and cndospcrm bcing ol morc unilorm color than ycllow corn kcrncls
with a whitc gcrm. 8ccausc ycllow corn absorbs morc light at 00nm, asymptomatic ycllow corn kcrncls
can bc distinguishcd lrom whitc corn kcrncls.
Acknowlcdgcmcnt:
Tis rcscarch was supportcd, in part, by thc Tcxas Corn Produccrs 8oard.
Maize LOX3 Gene is Required for Fumonisin Biosynthesis and Conidiation of
Fusarium verticillioides
Xiquan Gao
!
, Von8o Shim
!
, !vo Fcussncr
2
, and Mikc Kolomicts
!
!
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas;
2
Georg-
August-University Goettingen, Germany
!n this study wc tcstcd thc hypothcsis that 9lipoxygcnascs (9LXs) and thcir mctabolitcs arc
mycotoxin susccptibility lactors in corn that arc induccd and utilizcd by Fusarium verticillioides and
othcr phytopathogcnic lungi to incrcasc lungal sporulation and mycotoxin production in sccds. Tis
hypothcsis is bascd on thrcc kcy obscrvations: (!) oxylipins produccd lrom linolcic and othcr lrcc latty
acids in Aspergillus spp., so callcd psilactors, arc potcnt rcgulators ol sporogcncsis and mycotoxins
synthcsis, (2) thc primary products ol plant 9LX rcactions, latty acid hydropcroxidcs 9SHPT(),
which arc structurally similar to psilactors, strongly inducc both Aspergillus conidiation and mycotoxin
production in vitro, (1) transcript lcvcls ol a maizc 9LX gcnc, ZmLOX, arc induccd in corn lincs that
arc susccptiblc but not rcsistant to aatoxin contamination. To tcst our hypothcsis, wc gcncratcd maizc
ncarisogcnic lincs (N!Ls) that arc cithcr Mutatorinscrtional mutant or wild typc at thc ZmLOX
locus. Currcntly, mutants and wild typc N!Ls arc at thc 8CF stagc in 871 gcnctic background
which is susccptiblc to lumonisin contamination. xylipin pronling suggcstcd that gcrminating lox
mutants arc dcvoid ol most 9LX dcrivcd latty acid hydropcroxidcs. Fumonisin 8 production and
conidiation ol F. verticillioides wcrc drastically rcduccd whcn thc lungus was grown on mutant lox
kcrncls providing a strong support ol our hypothcsis. Morcovcr, conidiation ol a distantly rclatcd lungal
spccics, Colletotrichum graminicola, a causal agcnt ol anthracnosc lcal blight, was signincantly rcduccd
on lox mutant lcavcs as comparcd to wild typc lcavcs. !mportantly, lungal biomass ol both pathogcns
was not acctcd by thc lox mutation. Tcsc data strongly support our hypothcsis that 9LXdcrivcd
mctabolitcs positivcly rcgulatc both lungal conidiation and mycotoxin production and arc susccptibility
lactors in maizc.
Toxicity Responses of Corn to the Mycotoxin Fumonisin B
1
in the Absence of
Fusarium verticillioides Infection
A.M. Zimcri, L.. Villiams, R.T. Rilcy, and A.. Glcnn
USDA-ARS, R.B. Russell Research Center, Toxicology & Mycotoxin Research Unit, Athens, GA
Fusarium verticilliodes, thc causativc agcnt ol car rot in corn, produccs thc mycotoxin lumonisin 8
(F8), which is a potcnt compctitivc inhibitor ol ccramidc synthasc, a kcy cnzymc ncccssary lor
sphingolipid mctabolism. Consuming corn and corn products ladcn with F8 causcs a rangc ol spccics
spccinc discascs in animals and also has bccn shown to causc canccr in laboratory rodcnts. F8 may also
contributc to csophagcal canccr and ncural tubc birth dclccts in humans. Vhilc F8 contamination is
typically grcatcr whcn car rot damagc is morc scvcrc, rcccnt rcports havc indicatcd corn hybrids can
bc inlcctcd with Fusarium and contaminatcd with F8, yct do not always cxhibit phcnotypic signs
ol car rot. Vc hypothcsizc that brccding programs may havc inadvcrtcntly sclcctcd lor cndophytic
lungal associations whcrcby F. verticillioides inlccts corn but docs not causc scvcrc car rot, yct may still
contaminatc corn with signincant lcvcls ol F8. Vc havc bcgun to addrcss thc rolc that F8 may play
in thc ovcrall biology ol thc lungal association with corn and havc dcvclopcd an cxpcrimcntal systcm
bascd on obscrvations that F8producing strains ol F. verticillioides arc pathogcnic against sccdlings
ol susccptiblc corn hybrids whilc nonproducing strains causc no discasc symptoms. Numcrous corn
lincs wcrc scrccncd lor thcir scnsitivity or inscnsitivity to F8 to cvaluatc which phcnotypc was most
prcvalcnt within corn. Tis study was cnhanccd by a ncw stratcgy lor disinlcsting sccd both cxtcrnally
and intcrnally using chlorinc gas. Tc grcat advantagc with this tcchniquc was that sccd wcrc not
imbibcd and could thus bc stcrilizcd morc cmcicntly and storcd until nccdcd. Stcrilc sccd ol cach corn
linc wcrc placc on agar supplcmcntcd with various conccntrations ol F8 (0, !, !0, and !00 M). Altcr
7!0 days, root and shoot wcight (wct and dry) and lcngth wcrc notcd. Tcosintc and Tripsacum wcrc also
cvaluatcd lor thcir scnsitivity to F8 sincc tcosintc is thc likcly progcnitor ol modcrn corn and Tripsacum
is thc sistcr taxon to Zea. Rcsults indicatcd corn sccdlings wcrc in gcncral scnsitivc to F8, with tcosintc
and Tripsacum also bcing vcry scnsitivc. nly onc corn linc, V21, was vcry inscnsitivc to F8. !n both
scnsitivc and inscnsitivc corn lincs, low lcvcls ol F8 (! M) stimulatcd root and shoot growth. Scnsitivc
corn lincs had scvcrcly inhibitcd growth ol roots and grcatly rcduccd gcrmination ratcs whcn cxposcd
to highcr conccntrations ol F8 ( !0 M). V21 gcrminatcd and grcw wcll cvcn on !00 M F8. Tus,
data supportcd F8scnsitivity as anccstral whilc inscnsitivity ol corn to F8 toxicity may bc rcccntly
dcrivcd. Tough F8 alonc causcd stunting ol acrial tissucs and rcduccd root mass, it was not sumcicnt
to causc thc lull suitc ol sccdling blight discasc symptoms causcd by a Fusarium inlcction. To dctcrminc
il othcr sccondary mctabolitcs work syncrgistically with F8 to causc discasc, wc gcrminatcd sccdlings
in thc prcscncc ol lungal cxtracts with and without F8. Solvcnt cxtracts wcrc madc ol F8producing
and nonproducing strains grown on corn lor !4 days. Vc lound that sccdlings cxposcd to cxtracts
containing F8 cxhibitcd sccdling blight discasc as sccn in plants inlcctcd with wildtypc Fusarium.
Sccdlings cxposcd to cxtracts that did not contain F8 grcw similar to thc control plants. !n addition
to mcchanistic cxaminations ol F8 toxicity in scnsitivc and inscnsitivc sccdlings, luturc work also will
locus on whcthcr F8 is absorbcd and translocatcd throughout thc plant. Vc will also invcstigatc thc
impact ol F8 and othcr molcculcs on systcmic signaling within corn sccdlings.
PANEL DISCUSSION: Fumonisin Elimination
Panel Chair: Charlcs Voloshuk
Panel Members: Jamcs Holland, on Vicklow, Mikc Kolomicts, Annc Maric Zimcri
Tc pancl discussion bcgan with an announccmcnt by Voloshuk that thc proposal to scqucncc thc
gcnomc ol Fusarium verticillioides was lundcd. !t is anticipatcd that scqucncing and asscmbly will bc
complctcd by May 2006. Spccial apprcciation was givcn to thc USAARS Mycotoxin Rcscarch
Unit in Pcoria and Syngcnta. Tc Mycotoxin Rcscarch Unit madc public all thcir ST scqucnccs
and Syngcnta madc public thc gcnomic scqucncc data. Tcir actions providcd signincant support and
lcvcragc to thc proposal. Acknowlcdgcmcnt was also givcn to thosc who supplicd lcttcrs in support.
Voloshuk was askcd whcthcr thc commcrcial hybrids or imbrcd lincs wcrc uscd in his study. His
rcply was commcrcial hybrids (8ccks Hybrids). Voloshuk was askcd about pH changcs in kcrncls
ovcrtimc. His rcply was that hc did not prcscnt thc rcsults in his prcscntation, but pH was monitorcd
during thc study. Tc initial pH ol uninoculatcd kcrncls was vcry similar lor all kcrncl stagcs and
did not changc ovcr thc coarsc ol thc cxpcrimcnt. Colonization by thc wild typc lungus rcsultcd in
incrcascd pH in blistcr and milk stagcs, dccrcascd pH in dcnt and maturc stagcs, and littlc pH changc
in dough. Voloshuk was askcd about shilts in thc typcs ol latty acids that might occur during kcrncl
dcvclopmcnt. Voloshuk could not answcr this qucstion but commcntcd that most ol thc lipids was in
thc gcrm tissucs. Tcrc was no commcnt lrom thc othcr pancl mcmbcrs or thc audicncc. Voloshuk was
askcd about thc corrclation bctwccn rcsults obtain lrom kcrncl assay and ncld assays. Hc rcplicd that hc
bclicvcd thcrc should bc a corrclation. Hc notcd thc ncld studics by Gary Payncs group, who obscrvcd
naturally occurring kcrncl inlcctions as carly as thc milk stagc ol dcvclopmcnt.
Holland was askcd to clarily thc discrcpancy bctwccn thc vcry high cstimatcs ol gcnctic corrclations
bctwccn Fusarium car rot and lumonisin contcnt and thc idcntincation ol somc QTL that acct only
onc ol thc two traits. His rcply was that both mcthods involvc cstimation and may havc dicrcnt crrors
associatcd with thcm, so that thc discrcpancy is an artilact ol thc usc ol dicrcnt statistical tcchniqucs.
Hc proposcd to dircctly tcst thc hypothcsis that thc gcncs accting Fusarium car rot arc largcly thc samc
as thc gcncs accting lumonisin contcnt by sclccting against car rot in onc population and cvaluating
thc ccct ol sclcction on lumonisin contcnt. Tis study is undcrway.
Zimcri was askcd il lumonisin has bccn dctcctcd in thc soil without plant matcrial. Shc indicatcd
that Ron Rilcy was thc pcrson who pcrlormcd thc cxpcrimcnt to answcr this qucstion, and that did not
know il hc scparatcd thc plant matcrial lrom thc soil.
Vicklow was askcd about thc cost ol applying thc N!R grain sortcr. Hc rcplicd that thc commcrcial
highspccd dualwavclcngth sortcr (ScanMastcr!! 2000 , SatakcUSA, Houston, TX) thcy tcstcd
lor rcmoving whitc corn contaminatcd with lumonisin had a capacity ol 7000 kg pcr hour. Potcntial
usc would bc on high valuc grains or as a cost ccctivc mcthod lor salvaging good quality kcrncls lrom
grain lots rcjcctcd lor damagc and mycotoxin contamination.
Kolomicts was askcd about thc growth charactcristics ol his lox1 mutants. Hc indicatcd that whilc
plant hcight was rcduccd up to 10 ol thc wild typc, thc grain wcight and yicld lrom thc mutants was
not acctcd whcn grown in thc grccnhousc. Furthcr tcsting ol lox1 mutants will bc pcrlormcd in thc
ncld during ncxt growing scason. Kolomicts was also askcd about thc spccinc contcnt ol thc kcrncl oils.
Hc rcplicd that with limitcd sccd only gcrminatcd kcrncls wcrc tcstcd lor somc complcx lipids and no
signincant dicrcncc lrom wild typc wcrc dctcctcd. Futurc studics would answcr this qucstion.
QTL Mapping for Fusarium Ear Rot and Fumonisin Contamination Resistance
in Two Populations of Maize (Zea mays)
Lcilani A. Robcrtson
!,2
, Michacl P. Jincs
2
, Pctcr 8alintKurti
!,4
, Gary A. Paync
!
, onald G.
Vhitc
1
, and Jamcs 8. Holland
2,4
!
2
Department of Crop
Science, North Carolina State University, Raleigh, NC;
1
Department of Crop Sciences, University of Illinois,
Urbana-Champaign, IL;
4
USDA-ARS, North Carolina State University, Raleigh, NC
Fusarium verticillioides and F. proliferatum arc lungal pathogcns ol maizc that causc car rot and
contaminatc grain with lumonisins, a lamily ol mycotoxins that advcrscly accts animal and human
hcalth. Tc objcctivc ol this study was to idcntily QTL lor rcsistancc to Fusarium car rot and lumonisin
contamination in two maizc populations, compriscd ol 2! 8C
!
F
!:2
lamilics lrom thc nrst backcross ol
G440 to FR!064 (GFR) and !41 rccombinant inbrcd lincs lrom thc cross NC100 8!04 (NC8).
QTL mapping was uscd to invcstigatc whcthcr QTL wcrc consistcnt across populations and thc
gcnctic rclationships bctwccn rcsistanccs to car rot and to lumonisin contamination. !n thc GFR
population, six QTL cxplaincd 41.6 ol thc phcnotypic variation lor mcan car rot rcsistancc across
cnvironmcnts and ninc QTL with onc cpistatic intcraction cxplaincd 66.6 ol thc variation lor mcan
lumonisin conccntration across cnvironmcnts. !n thc NC8 population, nvc QTL cxplaincd 1!.1 ol thc
phcnotypic variation lor mcan car rot rcsistancc across cnvironmcnts and six QTL and thrcc cpistatic
intcractions cxplaincd 0.! ol thc phcnotypic variation lor mcan lumonisin conccntration across
cnvironmcnts. Trcc QTL in thc GFR population and lour QTL in thc NC8 population acctcd
both car rot and lumonisin conccntration. Trcc car rot and thrcc lumonisin contamination rcsistancc
QTL mappcd to similar positions in thc two populations. nc QTL localizcd to chromosomc 4
appcarcd to bc consistcnt lor both traits across both populations.
Polyketide Synthases in Fusarium verticillioides: Potential Targets to Control
Fumonisin Contamination in Corn
Robcrt H. Proctor, Robcrt A.. 8utchko, Ronald . Plattncr, Mark 8usman, arcn V. 8rown,
and Annc . csjardins
USDA, ARS, National Center for Agricultural Utilization Research, Peoria, IL
Tc lungus Fusarium verticillioides ncgativcly impacts corn production in two ways, it produccs thc
mycotoxins lumonisins and it causcs discascs such as car and stalk rot. Fumonisins can causc a numbcr ol
animal discascs, including canccr and ncural tubc dclccts in laboratory rodcnts. !n humans, consumption
ol lumonisincontaminatcd corn has bccn corrclatcd cpidcmiologically with csophagcal canccr and
ncural tubc dclccts. Sorting ol individual kcrncls lrom F. verticillioidesinoculatcd corn rcvcalcd that
lumonisin lcvcls arc substantially highcr in symptomatic kcrncls comparcd to asymptomatic kcrncls
(csjardins & Plattncr, Plant is. !998, 82: 9198). Tis nnding indicatcs that a rcduction in car rot
should rcducc lumonisin contamination in corn. Tus, a goal ol our rcscarch is to idcntily lactors that
contributc to thc ability ol F. verticillioides to causc car rot bccausc such lactors arc potcntial targcts lor
discasc control.
Production ol somc polykctidcdcrivcd mctabolitcs contributcs to thc ability ol a numbcr ol
lungi (c.g. Cercospora, Cochliobolus and Phylosticta) to causc plant discasc. Tcrclorc, polykctidcs may
contributc to thc ability ol F. verticillioides to causc corn car rot. Polykctidc synthascs (PKSs) typically
catalyzc an carly stcp in thc biosynthcsis ol polykctidcs, and disruption ol a PKS gcnc blocks production
ol thc corrcsponding polykctidc(s). Filtccn PKS gcncs havc bccn idcntincd in thc F. verticillioides
gcnomc (Krokcn ct al., Proc. Nat. Acad. Sci. USA 2001, !00: !670!67). To dctcrminc thc rolc ol
F. verticillioidesproduccd polykctidcs in pathogcncsis, wc arc disrupting cach PKS gcnc. To datc, wc
havc disruptcd cight ol thc PKS gcncs and dctcrmincd that onc (PKS) is rcquircd lor production ol
thc mycotoxins lusarins (also shown by Song ct al., Chcm8ioChcm 2004, : !!96!201) and anothcr
(PKS) is rcquircd lor production ol thc dark pigmcnt in thc walls ol thc scxual lruiting bodics ol F.
verticillioides. Pathogcnicity tcsts with all cight PKS mutant strains arc in progrcss.
Prcvious Northcrn blot analysis indicatc thc nltccn gcncs in lumonisin biosynthctic (FUM) gcnc
clustcr, including thc PKS gcnc FUM, cxhibit similar pattcrns ol cxprcssion. Tis cocxprcssion ol
FUM gcncs was also dctcctcd by microarray analysis ol ovcr !!,000 F. verticillioides ST scqucnccs.
8ascd on thcsc rcsults, wc arc using microarray analysis to cxaminc thc cxprcssion pattcrns ol thc F.
verticillioides PKS gcncs and thcir anking gcncs to idcntily polykctidc biosynthctic gcnc clustcrs.
Prcliminary analysis suggcsts that two PKS gcncs, PKS and PKS, arc part ol polykctidc biosynthctic
gcnc clustcrs. Tc cight contiguous gcncs on onc sidc ol PKS cxhibit cocxprcssion whilc thc gcncs
on thc othcr sidc do not. Scqucncc comparisons indicatc that six ol thc cocxprcsscd gcncs cncodc
cnzymcs (c.g. oxidorcductascs and a carboxymcthly translcrasc) consistcnt with thc prcdictcd lusarin
biosynthctic pathway. Tc scvcn contiguous gcncs adjaccnt to PKS also cxhibit cocxprcssion.
Computational Studies on the Infuence of Solvent on the Conformational
Preferences and Selective Recognition of Fumonisins
M. Appcll, C.M. Maragos, and .F. Kcndra
USDA-ARS, National Center for Agricultural Utilization Research, Peoria, IL
Sclcctivc rccognition is an important lactor in thc dcvclopmcnt ol matcrials to bind and dctcct
mycotoxins, including lumonisins. At thc molccular lcvcl, sclcctivc rccognition can bc modclcd as thc
intcraction ol thc mycotoxin with thc binding matcrial. Tis typc ol molccular rccognition is highly
dcpcndcnt on thc conlormation ol thc mycotoxin and thc binding sitc. !n addition, solvcnt has an ccct
on conlormations ol thc intcracting spccics and thc binding intcractions. !n our computational studics,
wc idcntincd scvcral stablc conlormations lor lumonisins A, A, 8, 8, and 8 in cxplicit watcr and
in vacuo. Calculations ol thc prclcrrcd conlormations ol lumonisins intcracting with potcntial binding
sitcs will providc usclul inlormation lor thc dcsign ol lumonisin sclcctivc binding matcrials.
Using Genomics Approaches to Characterize Potential Fumonisin Regulatory
Genes
Robcrt A.. 8utchko
!
, Robcrt H. Proctor
!
, arcn V. 8rown
!
, Charlcs P. Voloshuk
2
, 8urton
H. 8luhm
2
and Mark 8usman
!
!
Mycotoxin Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL;
2
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN
Fusarium verticillioides can causc sccdling discasc, stalk rot and car rot ol maizc and also produccs thc
mycotoxins lumonisins. Fumonisins arc polykctidc dcrivcd sccondary mctabolitcs synthcsizcd through
a multistcp biosynthctic pathway by cnzymcs cncodcd by a corcgulatcd clustcr ol gcncs (FUM gcnc
clustcr). Fumonisins arc toxic to both humans and animals and havc most rcccntly bccn dcscribcd
as tcratogcnic, causing ncural tubc dclccts in micc. !n an cort to rcducc or climinatc lumonisin
contamination ol maizc wc arc cmploying gcnomic rcsourccs to clucidatc thc gcnctic rcgulation
ol lumonisin production. Trcc typcs ol F. verticillioides gcnomic rcsourccs arc availablc: cxprcsscd
scqucncc tag (STs) librarics, microarrays and wholc gcnomc scqucncc.
Vc havc dcvclopcd an ST library containing ovcr 87,000 scqucnccs, in collaboration with Tc
!nstitutc lor Gcnomic Rcscarch (T!GR), which rcprcscnts !!,!!9 dicrcnt scqucnccs. Tc cNA
librarics wcrc constructcd lrom mRNAs isolatcd lrom cight dicrcnt growth conditions. Tc publicly
acccssiblc T!GR F. verticillioides Gcnc !ndcx (FvG!) incorporatcs all availablc scqucncc data into onc
databasc and at prcscnt, includcs !!,!26 total uniquc scqucnccs. Vc havc utilizcd thc ST librarics
to idcntily possiblc rcgulatory gcncs. Comparison ol librarics lrom conditions whcrc thc FUM gcnc
clustcr is not transcribcd and conditions whcrc thc FUM gcnc clustcr is transcribcd has allowcd thc
idcntincation ol a numbcr gcncs with similarity to rcgulatory typc gcncs which may havc a rolc in thc
transcriptional rcgulation ol thc FUM gcnc clustcr. Vc havc disruptcd a numbcr ol thcsc candidatcs,
six ol which havc an ccct on thc transcription ol thc FUM gcncs.
Vc havc also gcncratcd a NimblcGcn oligonuclcotidc microarray, in collaboration with T!GR, bascd
on thc FvG!. Tc microarrays consist ol approximatcly !80,000 24basc pair probcs or lcaturcs, with
cach scqucncc in thc FvG! rcprcscntcd by a sct ol !2 probcs. Vc havc validatcd thc microarrays using
mRNA gcncratcd lrom wildtypc F. verticillioides culturcd on lumonisininducing mcdia. mRNA was
isolatcd at 6 timc points ovcr days and was uscd to probc thc microarrays. FUM gcncs cxhibitcd
pattcrns ol cxprcssion cxpcctcd bascd on prcvious Northcrn analysis. Analysis ol this timc coursc
cxpcrimcnt will allow us to sct basclinc lcvcls ol cxprcssion across thc sct ol gcncs rcprcscntcd on thc
chip lor comparison in othcr cxpcrimcnts. Vc arc currcntly invcstigating dicrcntial gcnc cxprcssion
bctwccn wildtypc F. verticillioides and a FCK mutant culturcd on wholc maizc kcrncls, as wcll as on
disscctcd cndospcrm and gcrm tissuc.
Rcccntly, 4X covcragc ol thc Fusarium verticillioides gcnomc gcncratcd at Syngcnta and asscmblcd
at thc 8road !nstitutc was madc availablc to thc public. Tc intcrscction ol wholc gcnomc scqucncc,
ST librarics and microarrays is allowing us to morc comprchcnsivcly dcnnc gcncs and dcscribc thcir
cxprcssion at thc transcription lcvcl.
Fumonisins in Maize in Guatemala, Preliminary Exposure Estimate, and
Policies and Recommendations to Minimize Exposure
Ronald T. Rilcy
!,
, lga A. Torrcs
2
, dwin Palcncia
2
, L. Lopcz dc Pratdcsaba
2
, Anthony. .
Glcnn
!
, Kcrry onncll
1
and Mario Fucntcs
4
!
Toxicology and Mycotoxin Research Unit, R. B. Russell Agricultural Research Center USDA-ARS, Athens,
GA;
2
Instituto de Nutricion de Centro America Y Panama, Guatemala, Central America;
1
Microbial Genomics
and Bioprocessing Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL;
4
Institute of Agricultural Science and Technology, Guatemala, Central America
From 20002001, maizc samplcs wcrc collcctcd lrom nclds in thc highlands (> !60 m) and lowlands
(< 160 m) ol Guatcmala. Tc rcsults showcd that maizc grown in thc lowlands had signincantly highcr
lcvcls ol lumonisins than thc maizc grown in highlands. Approximatcly 92 ol thc samplcs lrom thc
lowlands collcctcd at harvcst in 2002 containcd dctcctablc lcvcls ol F8, whcrcas, only ol thc samplcs
collcctcd at harvcst lrom thc highlands containcd dctcctablc lumonisins. Howcvcr, 27 ol samplcs ol
thc 2002 crop collcctcd lrom storagc in thc highlands immcdiatcly bclorc harvcst ol thc 2001 crop
containcd 0.1 ppm F8 comparcd to only 2 ol thc samplcs collcctcd at harvcst in 2002. All (!00)
ol thc Fusarium inlcctcd kcrncls (60/!80) analyzcd lrom ninc random lowland samplcs (20 kcrncls/
samplc) wcrc inlcctcd with F. verticillioides (60/60) and no othcr Fusarium spccics, whcrcas, in samplcs
lrom thc highlands (n - 9) only (2/41) ol thc Fusarium positivc kcrncls (41/!80) wcrc F. verticillioides.
All thc F. verticillioides isolatcs wcrc ablc to producc lumonisin in culturc. !n FY 200 maizc samplcs
(n - 216) lrom thc 2004 crop wcrc analyzcd lrom highland and lowland markcts in Guatcmala. Tc
rcsults show that lowland maizc, highly contaminatcd with lumonisin, is sold in highland markcts
in cpartmcnts whcrc thc incidcncc ol ncural tubc dclcct is somctimcs vcry high. Tus, lumonisin
cxposurc in high risk arcas will bc grcatcst in groups that obtain thcir maizc lrom thc markct placc sincc
wc havc shown that maizc that is grown in thc highlands contains vcry low lcvcls ol lumonisins. 8ascd
on a rccall study in womcn conductcd in thc Ccntral Highlands, a prcliminary asscssmcnt ol daily
intakc ol total F8s was cstimatcd. Consumption ol nixtamalizcd maizc products madc lrom lowland
maizc could rcsult in cxposurc cxcccding thc provisional maximal tolcrablc daily intakc (2 g total
lumonisins/kg bw) with ovcr 0 ol thc maizc samplcs. Policics and rccommcndations to minimizc
lumonisin cxposurc in Guatcmala havc bccn discusscd. Tcsc rccommcndations arc intcndcd to cstablish
a prudcnt public hcalth policy that will minimizc risks to human hcalth whilc also minimizing ncgativc
impacts on thc maizc industry. Tcy can bc achicvcd through thc usc ol good agricultural and good
proccssing/cooking practiccs and cducation ol high risk populations and hcalth providcrs in Guatcmala.
Tis work was supportcd by USA Forcign Agricultural Scrvicc grant X0!4!0627!07!4, a grant
lrom thc !LS! NA Tcchnical Committcc on Food Toxicology and Salcty Asscssmcnt and support lrom
thc !nstituto dc Nutricion dc Ccntro Amcrica Y Panama.
Fusaric Acid, a Fusarium verticillioides Miasma to Bacillus mojavensis, a Biological
Control Bacterial Endophyte
Charlcs V. 8acon and . M. Hinton
USDA, ARS, Russell Research Center, Toxicology & Mycotoxin Research Unit, Athens, GA
Antagonisms among microorganisms arc stratcgics that maintain both intcr and intra spccinc
compctition, which is particularly important among thosc microorganisms that arc ccological
homologucs. Fusarium verticillioides is systcmically localizcd in corn and is prcvalcnt in thc roots as
opposcd to thc shoot axis ol corn, and is bcst dcscribcd as a root cndophytc. uring its carly biotrophic
phasc ol its association with corn, hyphac dwcll within thc intcrccllular spaccs ol corn. A biocontrol
bactcrium, Bacillus mojavensis, is patcntcd as an cndophytic biocontrol agcnt ol plant discascs. Tc
intcntion is to rcplacc thc lungus with this cndophytic bactcrium as a managcmcnt stratcgy which
opcratcs undcr thc broad mcchanism ol compctitivc cxclusion. Undcr grccnhousc conditions, corn
inlcctcd with thc bactcrium shows incrcascd growth and rooting, sccdling vigor and discasc rcsistancc.
Also undcr thcsc conditions, lumonisin conccntration in corn is rcduccd by 60 in thc prcscncc ol
thc lungus, and lungus inlcction, cxprcsscd as CFU/gram ol plant tissuc, is also rcduccd. Howcvcr,
usc ol this bactcrium undcr ncld conditions and contrary to grccnhousc conditions, F. verticillioides is
supcrior in colonizing corn plants prcinoculatcd with thc bactcrium. l thc many toxins produccd
by F. verticillioides, lusaric acid might bc involvcd in this ccct. Fusaric acid (butylpicolinic acid),
nrst discovcrcd during thc laboratory culturc ol F. heterosporum, was onc ol thc nrst lungal mctabolitcs
implicatcd in thc pathogcncsis ol wilt symptoms ol plants. !n addition to this rolc in plant pathogcncsis,
lusaric acid is mildly toxic to micc, and has scvcral important pharmacological propcrtics, and
pcrhaps its major importancc in animal toxicity may bc syncrgistic intcractions with othcr naturally
cooccurring mycotoxins. !t was dctcrmincd that lusaric acid accountcd lor thc rcduction in bactcrial
growth and rcsulting dccrcasc in biocontrol activity. Fusaric acid supplicd to culturcs ol thc bactcrium,
at a conccntration as low as 22 M, accountcd lor a 4! rcduction in growth ol thc bactcrium. !t is
also toxic to this bactcrium. Fusaric acidlcss mutants ol F. verticillioides wcrc inccctivc in colonizing
B. mojavensisinlcctcd maizc, suggcsting that lusaric acid is onc important dclcnsc mcchanism lor thc
lungus. Tc rcsults indicatc that thc biocontrol bactcrium must bc modincd to rcsist lusaric acid bclorc
its usc undcr ncld conditions. Two lusaric acid tolcrant bactcrial mutants havc bccn dcvclopcd that arc
cndophytic and antagonistic to thc lungus. Tcsc mutants will lorm thc basis ol subscqucnt ncld tcsting
lor thc control ol F. verticillioides.
Developmental Toxicity of Fusarium verticillioides and Fumonisin B in LM/Bc
and CD1 Mice: Comparing the in vivo Models
Kcnncth A. \oss
!
, Ronald T. Rilcy
!
, Tantiana . 8urns
!,2
& Jancc 8. Gclincauvan Vacs
1
!
Toxicology & Mycotoxin Research Unit, RB Russell Agricultural Research Center USDA-ARS, Athens, GA;
2
Interdisciplinary Toxicology Program, University of Georgia, Athens, GA;
1
Department of Genetics, Cell
Biology & Anatomy, Omaha, NE
Tc human hcalth cccts ol Fusarium verticillioides and lumonisins arc unccrtain. Tcrc is cvidcncc
howcvcr suggcsting that lumonisins disrupt lolatc utilization and incrcasc thc risk ol ncural tubc dclccts
(NTs - birth dclccts causc by lailurc ol thc ncural tubc to closc propcrly) in populations that dcpcnd
hcavily on lumonisincontaminatcd corn as a lood sourcc. Fumonisin 8 (F8) was not tcratogcnic
whcn givcn orally (gavagc) to prcgnant C! micc on gcstation days (G) 7! whcrcas intrapcritoncal
(ip) injcction ol mg/kg 8V F8 on G7 and G8, thc critical timc lor ncural tubc closurc, to
prcgnant LM/8c micc causcd NTs. xpcrimcnts wcrc thcrclorc donc to comparc thc incidcncc
ol NTs in littcrs ol LM/8c and C! dams givcn F8 by two dicrcnt dosing protocols: (a) dictary
cxposurc to lumonisins (providcd by adding F. verticillioides culturc matcrial to thc dict) bcginning
wccks bclorc mating and (b) ip administration ol F8 on G7 and G8.
Tc rcsults ol thc lccding studics wcrc inconclusivc. icts containing 0 ppm F8 did not causc
NTs in cithcr strain. At thc matcrnally toxic dosc ol !0 ppm F8, onc ol nvc LM/8c littcrs was
NT positivc (!/!0 lctuscs acctcd) whcrcas lctal dcath ratcs wcrc highcr but no NTs wcrc lound in
thc C! strain (n - 9 littcrs). !n a sccond lccding trial using LM/8c micc, NTs wcrc not lound in thc
lctuscs ol lcmalcs lcd dicts containing !0 or 100 ppm F8.
A doscrclatcd incrcasc in NTs was lound in thc littcrs ol C! dams (n - 8!0/dosc lcvcl) givcn
F8 by ip injcction on G7 and G8: 0, !!, 0, and 40 pcrccnt ol thc littcrs wcrc NT positivc at doscs
ol 0, !, 10 and 4 mg/kg 8V F8, rcspcctivcly. Tis rcsult was connrmcd in a sccond cxpcrimcnt.
NTs wcrc lound in 0, 8.1, !6.6, 16.4, 4. pcrccnt ol thc littcrs ol C! dams (n - 8!2/dosc lcvcl) givcn
0, !0, 21, 4 or !00 mg F8/kg 8Vt F8 ip on G7 and G8. !n acctcd littcrs ol dams givcn 4
ppm F8, 11 pcrccnt or lcss ol thc C! lctuscs had NTs. Tc numbcr ol NT positivc lctuscs lrom
acctcd littcrs ol C! dams givcn !00 mg/kg 8Vt F8 tcndcd to bc highcr: ! to !00 pcrccnt cxhibitcd
NT (avcragc mcan lor thc group - 42 pcrccnt). !n contrast, !00 pcrccnt ol thc littcrs and 0 pcrccnt
ol thc lctuscs lrom LM/8c dams givcn ! mg/kg F8 by this ip dosing schcdulc wcrc NT positivc.
Tcsc rcsults indicatc that (a) both mousc strain and dosing rcgimcn acct NT induction, (b)
induction ol NTs by ip F8 cxposurc during thc critical timc lor ncural tubc closurc is not uniquc to
thc LM/8c mousc strain, (c) LM/8c micc arc morc scnsitivc to NT induction than C! micc, and (d)
uncquivocal induction ol NTs by dictary cxposurc to lumonisins rcmains to bc shown. Comparativc
studics using lumonisincxposcd LM/8c and C! micc will bc usclul lor clucidating thc physiological
and biochcmical cvcnts involvcd in NT lormation in vivo.
64
+8:n Axxu~i Avi~:oxix ii:ix~:iox Vovxsnov: Svssiox + 65
18
TH
SESSION 1: CROP RESISTANCE CONVENTIONAL BREEDING
Moderator: on Joncs, Cotton !ncorporatcd
64
Creation of Commercial Hybrids with Low Afatoxin in Grain using Markers
on Vhitc and Torbcrt Rochclord
Department of Crop Sciences, University of Illinois, Urbana, IL
Tis projcct is crcating high yiclding, commcrcially acccptablc, corn hybrids with high lcvcls ol
rcsistancc to Aspcrgillus car rot and low lcvcls ol aatoxin in grain. Tis is bcing accomplishcd by using
molccular markcr assistcd backcrossing to movc chromosomc rcgions associatcd with rcsistancc lrom
rcsistant inbrcds Tcx6 and Mp1!1 into thc commcrcially clitc, but susccptiblc, inbrcd lincs FR!064
and LH!9RR.
Vith FR!064 wc havc crosscd FR!064 with Mp1!1 and back crosscd thrcc or lour timcs to FR!064
whilc sclccting lor thc chromosomc lour rcgion lrom MP1!1 that has bccn associatcd with rcsistancc.
Vc now havc nvc inbrcd lincs at thc scll scvcn lcvcl ol inbrccding that havc thc chromosomc rcgion
lour lrom Mp1!1. Tcsc lincs, at various stagcs ol backcrossing and sclnng, havc bccn cvaluatcd lor
aatoxin production and yicld as tcst cross hybrids with scvcral inbrcds including FR414! at locations
in Tcxas, Mississippi, !llinois and thc various locations providcd by SRAT. !n gcncral, in cxpcrimcnts
whcrc dicrcnccs occur, thc back crosscd rcsistant inbrcds havc 0 to 90 lcss aatoxin in tcst cross
hybrids than FR!064 in comparablc tcst crosscs. Although thc rcsistant lincs with chromosomc lour
lrom Mp1!1 arc approximatcly 94 similar to FR!064 thcy arc latcr in maturity, havc bcttcr husk
covcragc, and bcttcr yicld in cxpcrimcnts donc in Tcxas.
Vc also arc pyramiding thc chromosomc rcgion lrom Mp1!1 with chromosomc rcgions lrom Tcx6
associatcd with rcsistancc. Tcsc lincs arc much dicrcnt than thosc with just thc MP1!1 chromosomc
lour crosscd into FR!064. For thosc lincs wc took a vcrsion ol MP1!1 crosscd with FR!064 and back
crosscd twicc to FR!064 with chromosomc lour lrom MP1!1 and crosscd it with a linc that was
dcvclopcd lrom thc cross ol 871xTcx6 thcn backcrosscd to 871 and scllcd that had rcsistancc lrom
Tcx6 on chromosomcs 8,!0,2, and . Tcrclorc, thc pyramid lincs havc both 871 and FR!064 which
contributc yicld and agronomic charactcrs and both MP1!1 and Tcx6 chromosomc rcgions associatcd
with rcsistancc. Tc rcsulting lincs arc latcr in maturity than rcsistant vcrsions ol FR!064. Tcy also
havc grcatcr plant hcight and morc drought rcsistancc. Vhcn cvaluatcd as tcst cross hybrids thcsc lincs
usually havc lcss aatoxin in grain than comparablc tcst cross hybrids with FR!064. !n somc cxpcrimcnts
thcy havc bcttcr rcsistancc than thc rcsistant lincs with just chromosomc lour lrom Mp1!1. Howcvcr,
in othcr cxpcrimcnts thcy arc similar or highcr in aatoxin. Tcy havc dcmonstratcd thc highcst lcvcl ol
rcsistancc whcn conditions wcrc cxtrcmcly lavorablc lor aatoxin production.
Vc also arc using molccular markcrs to backcross rcsistancc lrom MP1!1 on chromosomc lour into
LH!9RR. Vc havc backcross two or thrcc vcrsions sclcctcd lor chromosomc lour. Vc nccd to makc six
backcrosscs to LH!9 in ordcr to ccctivcly rccovcr thc agronomic charactcrs ol LH!9 which is vcry
widcly uscd in corn hybrids lrom southcrn !llinois to thc dccp South.
Vith guidancc lrom Quinton Raab ol 8H Gcnctics wc havc conccntratcd on idcntilying potcntial
malc parcnts to bc uscd with thc rcsistant lcmalc parcnts that wc arc dcvcloping. !nbrcd lincs rclatcd
to Sti Stalk Synthctic such as FR!064, 871 and LH!9RR arc usually uscd as lcmalc parcnts ol
commcrcial hybrids bccausc ol good sccd quality and rapid sccdling cmcrgcncc. Tc malc parcnts that
wc havc idcntincd as contributing good yicld in hybrid combinations with our rcsistant lcmalc parcnts
includc FR6942, LH26 and TR9128t. Tcsc inbrcds arc widcly uscd as malc parcnts in corn hybrids
lrom Ccntral !llinois to thc dccp South. Tis summcr wc produccd commcrcial bag quantitics ol somc
ol our rcsistant inbrcds with LH26 and TR912. 8H Gcnctics is producing bag quantitics ol crosscs
with FR6942 in Florida this wintcr. Tc sccd will bc proccsscd and sccd trcatcd so that largc yicld
trials can bc conductcd in 2006. Vc also arc incrcasing sccd ol rcsistant lincs in Hawaii this wintcr
so that cvcn grcatcr amounts ol commcrcial sccd can bc produccd in 2006. Vc also will bc cvaluating
additional malc lincs with rcsistancc that havc bccn dcvclopcd by Javicr 8ctrn. Crosscs bctwccn two
rcsistant inbrcds may wcll rcsult in hybrids with grcatcr lcvcls ol rcsistancc.
66 +8:n Axxu~i Avi~:oxix ii:ix~:iox Vovxsnov: Svssiox +
Breeding Corn Germplasm for Agronomic Performance and Reduced Afatoxin
Contamination
Javicr 8ctrn, Tom !sakcit, Gary dvody, and Kcrry Mayncld
Texas A&M University, College Station, TX
ur program has cvaluatcd, idcntincd and dcvclopcd corn inbrcds with rcsistant lactors that can
rcducc thc risk ol aatoxin and havc a good agronomic pcrlormancc in hybrids. Vc havc uscd thrcc
locations in South Ccntral Tcxas and inoculation with Aspergillus favus (isolatc NRRL117) using
thc nonwounding silk channcl or colonizcd corn kcrncls on thc soil surlacc. At harvcst, inlcctcd cars
wcrc huskcd, ratcd lor kcrncl intcgrity and visiblc lungi colonization, shcllcd, ground with a mill, and
cvaluatcd lor aatoxin. Quantincation ol aatoxin was conductcd with monoclonal antibody amnity
columns and uorcsccncc dctcrmination (\icam Aatcst). Tcsc cxpcrimcntal scrccning tcchniqucs
and inoculation havc lacilitatcd thc display ol gcnctic dicrcnccs among inbrcds and hybrids, and
incrcascd hcritability in aatoxin cvaluations. Multiycar and multilocation tcsting has pcrmittcd thc
cstimation ol how much is thc gcnotypc by cnvironmcntal intcraction in aatoxin accumulation and thc
idcntincation ol whitc and ycllow lincs with thc most consistcnt rcsistancc. Tc rcplicatcd cvaluation
ol lincs and hybrids in scvcral locations and ycars was instrumcntal to idcntily gcnotypcs with thc bcst
rcsponsc in dicrcnt cnvironmcnts. !nbrcds CML121, Tx772, CML288, NC100, FR2!28, CML118,
CML!6! and cxpcrimcntal lincs TxX69s and TxLAMA among thc ycllows, and inbrcds CML!76,
CML269, CML78, and Tx807, and Tx cxpcrimcntal lincs dcrivcd lrom crosscs among CML269, Tx!!0,
CML78, and CML270 among thc whitcs arc thc most promising lincs to contributc rcsistant lactors
to aatoxin. Most ol thcsc lincs havc subtropical or tropical origin, an indication that cxotic gcrmplasm
can harbor gcncs that can contributc to rcducc thc risk ol aatoxin. !n addition, thcsc cxotic lincs havc
shown good combining ability and agronomic pcrlormancc in crosscs with tcmpcratc adaptcd inbrcds
LH!9 and LH2!0. Somc Argcntinc commcrcial hybrids (c.g., AX889 and Condor) havc also shown
lcss aatoxin conccntrations than U.S. commcrcial hybrids. Low aatoxin accumulation was associatcd
with good husk covcragc, inty cndospcrm tcxturc, and good kcrncl intcgrity. !t sccms plausiblc to
sclcct lor associatcd traits having high hcritabilitics and strong corrclation with aatoxin to rcducc thc
risk ol aatoxin contamination. arly maturing hybrids wcrc morc susccptiblc duc to lack ol adaptation,
bad husk covcragc and solt cndospcrm. Tc rclationship bctwccn aatoxin accumulation in inbrcds and
thcir hybrids has bccn variablc. Tc corrclation bctwccn inbrcd and hybrids havc bccn ol low prcdictivc
valuc in somc cxpcrimcnts and high in othcrs. Tc typc ol gcrmplasm cvaluatcd, thc numbcr ol lincs,
and thc gcnctic variation prcscnt has inucnccd this rclationship. Tc usc ol gcnctic dcsigns such as
diallcls, lactorial dcsigns and gcncration mcans analysis whcrc lincs arc cvaluatcd in scvcral crosscs havc
lacilitatcd thc idcntincation ol thosc lincs that pcrlorm bcttcr across hybrid combinations. Rccombinant
inbrcd linc (R!L) populations havc bccn dcvclopcd lrom sclcctcd lincs (c.g., CML!76 and CML!6!)
to map potcntial gcnomic rcgions or QTLs associatcd with rcsponsc to aatoxin and othcr sccondary
traits. Tc combincd cvaluations lor aatoxin and agronomic pcrlormancc has lacilitatcd thc sclcction
lor adaptation, yicld potcntial, stability, and rcduccd aatoxin risk. Ultimatcly, wc aim to incorporatc
aatoxin rcsistant lactors into clitc gcnctic backgrounds suitablc to producc commcrcial hybrids.
Interaction Between A. favus Strains and Host Plant Genotypes Across
Environments and Years
Kcrry Mayncld, Tom !sakcit, Gary dvody, and Javicr 8ctrn
o intcractions occur bctwccn gcnctically dicrcnt isolatcs ol Aspergillus favus and dicrcnt gcnotypcs
ol maizc: Currcntly onc isolatc ol A. favus has bccn uscd lor inoculation in our trials, although isolatcs
ol this spccics arc known to cxhibit a rangc ol toxigcnic capacity at dicrcnt cnvironmcnts. ur objcctivc
was to dctcrminc il thcrc is intcraction bctwccn gcncticallydicrcnt isolatcs ol A. favus and scvcral
gcnotypcs ol maizc. Two cxpcrimcnts wcrc conductcd in 2004 and 200, onc with hybrids and onc with
inbrcds. Tc hybrid trial containcd lour commcrcial hybrids and lour TAMU cxpcrimcntal hybrids. Tc
inbrcd trial includcd two whitc inbrcds, two high lysinc inbrcds, and onc ycllow inbrcd. !nbrcds and
hybrids wcrc sclcctcd lor maturity and prcvious rcsponsc to aatoxin (AF). Tc hybrid trial was plantcd
at Collcgc Station, TX (CS), Vcslaco, TX (V) and Corpus Christi, TX (CC) in 2004 and at CS and
V in 200. Tc inbrcd trial was plantcd at CS and V both ycars. An alphalatticc ncld cxpcrimcntal
dcsign was uscd in thc hybrid trial, and a randomizcd complctc block dcsign in thc inbrcd trial, both with
lour rcps. Hybrids and inbrcds wcrc inoculatcd using thc silk channcl inoculation mcthod using isolatcs
L!, F!, ! (isolatcd lrom soil in a maizc ncld in San Patricio County, Tcxas) and NRRL117 (AF117).
!solatcs wcrc inoculatcd with in thc samc row and kcpt scparatc by marking individual inoculatcd
plants with colorcd tapc. Plots wcrc hand harvcstcd, shcllcd and ground prior to quantincation ol AF
using \icam Aatcst. ata analysis was conductcd using SAS Proc GLM. Signincant dicrcnccs
among gcnotypcs wcrc dctcctcd in both inbrcd and hybrid trials in 2004 and 200 at both locations.
Signincant dicrcnccs among thc dicrcnt isolatcs wcrc also obtaincd lor aatoxin conccntration.
Aatoxin conccntrations across cnvironmcnts pcr isolatc in inbrcds wcrc 202 ng g
' lor AF117, 121 ng

g
' lor F!, 206 ng g
' lor L!, and 06 ng g
' lor !. !n hybrids, aatoxin avcragcs wcrc !29 ng g
' lor
AF117, 170 ng g
' lor F!, !18 ng g
' lor L!, and !97 ng g
' lor !. !solatcs F! and ! produccd morc

aatoxin than commonly uscd isolatc AF117. Graphs showcd slight intcraction bctwccn gcnotypc and
isolatc, howcvcr, this intcraction was non signincant at any location or trial, and ncithcr across locations
and ycars. Signincant isolatc by cnvironmcnt intcractions wcrc dctcctcd in thc hybrids both ycars and
across cnvironmcnts. !solatc F! produccd morc aatoxin in CS in 200, AF117 in CC in 2004, L! in
CS both ycars, and ! in CC in 2004. nc isolatc ol A. favus may bc uscd in scrccning lor rcsistancc,
howcvcr, rcsults may bc variablc in ycars that cnvironmcntal conditions arc unlavorablc lor that isolatc.
A mixturc ol local isolatcs may cnsurcd morc consistcnt aatoxin conccntrations to dicrcntiatc among
tcsting maizc gcnotypcs.
Application of HACCP to Control Mycotoxins in Maize Breeding Programs
avid F. Kcndra
Mycotoxin Research Unit, USDA, ARS, National Center for Agricultural Utilization Research, Peoria, IL
Maizc car rot and associatcd mycotoxin contamination arc scrious problcms lor maizc growcrs around
thc world. !n thc U.S. cornbclt scvcrc car rot and mycotoxin outbrcaks occur sporadically whilc thcy
arc scrious problcms ycarly in othcr rcgions such as thc southcastcrn U.S. uring hybrid sclcction,
commcrcial maizc brccdcrs routincly discard gcnotypcs that arc visibly susccptiblc so commcrcial
hybrids arc gcncrally somcwhat rcsistant to car rot, howcvcr, littlc inlormation is availablc on mycotoxin
rcsistancc lcvcls lor commcrcial maizc hybrids. uc to incrcascd public conccrn ovcr lood salcty and
its rolc in tradc policy dcvclopmcnt and ncgotiations, mycotoxins arc now morc closcly monitorcd with
at lcast 99 countrics having omcial rcgulations lor lood and/or lccd. uring thc last thrcc dccadcs,
thc Hazard Analysis Critical Control Point (HACCP) systcm has bccn gradually introduccd and
applicd succcsslully by thc lood industry to introducc risk asscssmcnt bascd cvaluations lor potcntial
contamination ol lood products with pathogcnic microorganisms and physical and chcmical
salcty hazards, including mycotoxins. HACCP is a proactivc, highly structurcd, systcmatic quality
managcmcnt systcm that includcs thc idcntincation, cvaluation and control ol hazards in thc cntirc
agricultural systcm. As a rcsult ol thc incrcascd importancc ol mycotoxins in global tradc, this papcr
rccommcnds that corn brccdcrs implcmcnt a HACCP bascd approach to dcvclop hybrids that mcct
or cxcccd intcrnational rcgulations lor rcgulatcd mycotoxins in ordcr to cnsurc compctitivcncss ol U.S.
larmcrs in thc global markct.
Characterizing Components of Insect-Based Resistance to Preharvest Afatoxin
Contamination in Almond
T.M. Gradzicl and A.M. andckar
Plant Sciences Department, University of California, Davis, CA
Aatoxin contamination in almond is strongly associatcd with inscct damagcd kcrncls. Prcharvcst
damagc is causcd primarily by thc naval orangcworm (Amyelois transitella) whilc postharvcst inlcstations
involvc both naval orangcworm and thc !ndian mcal moth. !ncorporating rcsistancc to thcsc insccts
complcmcnts corts to dcvclop aatoxin rcsistant varictics and allows sclcction stratcgics which
typically dcmonstratc lcss cnvironmcntal variancc thcn plant discasc scrccning mcthods, allowing
highcr nnal hcritability. Trcc major componcnts ol inscct rcsistancc havc bccn succcsslully utilizcd in
brccding lor aatoxin rcsistancc: high shcllscal intcgrity, nonprclcrcncc/antibiosis, and toxicity. Tc
intcgrity ol thc almond shcllscal is dctcrmincd primarily by thc inncr cndocarp laycr, particularly
in thc rcgion adjaccnt to thc vascular bundlcs lccding thc ovulcs. !n addition to a high rcsponsc
tosclcction, this approach has allowcd thc dcvclopmcnt ol rcsistant gcnotypcs having kcrncl/shcll
ratios cxcccding 6. !n nonprclcrcncc/antibiosis, insccts show rcduccd prclcrcncc towards rcsistant
gcnotypcs and whcn inlcstation occurs, show longcr larval dcvclopmcnt timcs whcn lccding on
rcsistant gcnotypcs. Advanccd sclcctions dcmonstrating vcry low lcvcls ol ncld inlcstations and almost
total supprcssion ol postharvcst inlcstation havc bccn dcvclopcd. !nscct toxicity is achicvcd through
thc sclcction ol high amygdalin lcvcls. Sincc almond is a cyanogcnic spccics, inscct lccding will rcsult
in thc brcakdown ol amygdalin, lorming bcnzaldchydc and thc toxin cyanidc. High amygdalin lcvcls
in thc kcrncl and/or hull and sccdcoat havc provcn ccctivc in controlling inscct damagc ol maturc
nuts in thc ncld. 8ccausc amygdalin accumulation within thc kcrncl and/or sccdcoat occurs latc during
sccd maturation, dcvcloping nuts may bc susccptiblc to inscct inlcstation during carlicr maturation
stagcs. !ntcrmcdiatc lcvcls ol amygdalin in thc still dcvcloping lruit ol highamygdalin gcnotypcs
as wcll as lully maturc sccd ol intcrmcdiatcamygdalin gcnotypcs may show highcr lcvcls ol inscct
damagc. 8cnzaldchydc appcars to bc a powcrlul attractant to oviposition and lccding by thcsc insccts
and it is hypothcsizcd that highcr inlcstation rcsults lrom thc ability ol bcnzaldchydc to act as an
attractant at lcvcls too low lor cyanidc toxicity.
Genetic and Genomic Approaches to Improve Host Resistance to Preharvest
Afatoxin Contamination in Corn and Peanut
8.Z. Guo
!
, M. Luo
!,2
, H. Chcn
2
, P. ang
1
, A.. Coy
2
, M.. Krakowsky
4
, . avis
, V. Xu
6
,
X. Liang
7
, C. Holbrook
4
, R.. Lcc
2
, M. 8aushcr
1
, A. Culbrcath
2
, P. ziasAkins
2
, and Craig K.
Kvicn
2
!
2
University of Georgia, Tifton,
GA;
1
USDA-ARS, Ft. Pierce, FL;
4
University of Columbia, MO;

6
7
Guangdong Academy of Agricultural
Sciences, Guangzhou, China
Planthost rcsistancc is a highly dcsirablc tactic that can bc uscd to managc pcst problcms. Scrccning and
idcntincation ol crop plant gcrmplasm lor rcsistant traits lor crop improvcmcnt and molccular markcr
dcvclopmcnt will bring ncw gcnctic divcrsity into U.S. corn/pcanut gcmplasm. Using thc combination
ol gcnctic and gcnomic approachcs to clucidatc crop dclcnsc pathways and undcrstand thc rcsistancc
mcchanism and rcgulation will cnhancc gcnctic brccding lor bcttcr crop cultivars and improvcd discasc
rcsistancc. Corn inbrcd lincs, GTA!!, GTP2 and GTP6 sclcctcd lrom GTMAS:gk population,
arc in latc stagc ol tcsting and will bc rclcascd soon. Corn inbrcd lincs, A618 (carly, !00 days) and CY!
(!! days), arc sclcctcd lrom gcrmplasm providcd lrom Spain and China. Scvcral pcanut lincs arc also
sclcctcd with vcry low lungal colonization in thc laboratory and low aatoxin lcvcls in thc ncld cagc
studics (2 ycar). A pcanut linkagc mapping population has bccn dcvclopcd lrom Tilrunncr (rcsistancc
to TSV\ and lcal spots) GTC20 (low aatoxin and rcsistancc to bactcria wilt) and will bc uscd
linkagc map construction and QTL studics.
Maizc microarray, both cNA and oligo arrays, havc bccn uscd to study thc gcnc cxprcssion pronlcs
in rcsponsc to drought strcss and Aspcrgillus inlcction. Maizc lincs uscd in thcsc studics wcrc GT
A!!, Tcx6, A618, 871, M0!7, L0964, L0!0!6, Tcx20 and Tcx202. Tcn crosstalking gcncs havc bccn
idcntincd and will bc uscd in gcnc cxprcssion analysis among morc inbrcd lincs, hybrids and R!Ls.
Vc havc bccn dcvcloping ST databasc as tools and rcsourccs lor pcanut community to gain gcnomic
inlormation and knowlcdgc and discovcr NAmarkcrs and gcncs. Vith thc nrst batch ol STs
submission to public domainGcn8ank in 2001, wc uscd this inlormation to charactcrizc somc pcanut
transcripts in rcsponsc to pcanut lcal spot discasc and Aspcrgillus inlcction and drought strcss using
pcanut cNA microarray. Pcanut sccd STs will bc complctcd soon with ovcr 20,000 STs lrom 6
cNA librarics at R, R6, and R7 stagcs ol Tilrunncr and GTC20.
Progress Toward Identifying New Sources of Genetic Variation Associated with
Reduced Levels of Afatoxin Accumulation in Maize
Tomas 8rooks
!
, Matthcw Krakowsky
2
, V. Paul Villiams
!
, and Gary Vindham
!
!
USDA-ARS, Corn Host Plant Resistance Research Unit, Mississippi State, MS;
2
USDA-ARS, Crop Genetics
and Breeding Research Unit, Tifton, GA
Abstract not submittcd.
Proteomic Identifcation of Maize Cob Proteins that Potentially Confer
Resistance to Afatoxin
awn Luthc
!
, lga Pcchanova
!
, 8cla Pccthambaran
!
, Tibor Pcchan
2
, Susan 8ridgcs
1
, Lcigh
Hawkins
4
, Gary Vindham
4
and V. Paul Villiams
4
!
Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, MS;
2
Life Sciences and Biotechnology Institute, Mississippi State University, Mississippi State, MS;
1
Department
of Computer Science, Mississippi State University, Mississippi State, MS;
4
USDA-ARS Corn Host Plant
Resistance Research Unit, Mississippi State University, Mississippi State, MS
!n this study, wc dctcrmincd thc protcomc ol thc dcvcloping maizc cob and silks 2! days altcr silking.
Using 2 gcl clcctrophorcsis and Multi imcnsional Protcin !dcntincation Tcchnology (MudP!T),
wc idcntincd approximatcly !600 cob and 900 silk protcins. !n addition, wc comparcd thc cob protcomc
ol rcsistant (R Mp1!1) and susccptiblc (S SC2!2m) inbrcds and inoculatcd and uninoculatcd cars
using icrcntial !n Gcl lcctrophorcsis (!G). !G analysis rcvcalcd intcrcsting dicrcnccs in
thc protcin composition bctwccn R and S lincs. !n gcncral, R containcd morc antioxidant cnzymcs,
small hcat shock protcins and cnzymcs involvcd in phcnolic mctabolism, whcrcas thc S containcd morc
chitinascs and a dicrcnt sct ol protcin in phcnolic mctabolism. Tc scts ol protcins induccd at !0 and
1 days altcr inoculation also dicrcd considcrably bctwccn R and S cobs. Similar typcs ol rcsults wcrc
lound lor silk protcins. Tc protcomic approach will allow us to sclcct protcin and gcncs lor markcr
sclcctcd brccding programs, in addition to providing clucs about thc mcchanisms ol allatoxin rcsistancc
in dcvcloping cars.
Development of Field Based Techniques for Assessing Variability Among
Cotton Cultivars in Susceptibility to Afatoxin Contamination During the
Second Phase of Contamination
M.V. lscn
!
, P.J. Cotty
2
and S. Husman
1
!
Department of Plant Sciences,
2
USDA-ARS,
1
Cooperative Extension, e University of Arizona, Tucson,
AZ
!n Arizona, southcrn Tcxas and thc !mpcrial \allcy ol Calilornia, aatoxins arc always a conccrn in
cottonsccd uscd lor animal lccd. Aatoxin contamination ol cottonsccd occurs in two phascs, thc nrst
whcn A. favus inlccts dcvcloping bolls through wounds or cracks, and thc sccond whcn maturc sccd
is cxposcd to both conducivc tcmpcraturcs and moisturc. Rank cotton, dcnsc canopics, dcw, and latc
irrigations incrcasc thc scvcrity ol sccond phasc contamination. At prcscnt thcrc arc no assays availablc
to comparc susccptibility ol cotton cultivars to sccond phasc contamination undcr ncld conditions.
cvclopmcnt ol such an assay is thc nrst objcctivc ol this projcct. Anothcr objcctivc is to dctcrminc il
sccd hardncss and sccd coat lragility arc rclatcd to aatoxin contamination. clibcratc wctting ol opcn
bolls using a mist systcm is bcing uscd to simulatc moisturc cccts.
!n 2004 trials, bolls wcrc mistcd thrcc timcs lor onc day cach. Humidity was incrcascd ovcr non
trcatcd controls, but not to an acccptablc lcvcl, and aatoxin contamination ol sccd was vcry low to
nondctcctablc in all samplcs. !n 200 trials, two dicrcnt cxpcrimcnts wcrc donc. Tc nrst was a
continuation lrom 2004 ol invcstigations ol thc timing ol wctting on aatoxin contamination. Tcrc
wcrc lour trcatmcnts with cight rcplications cach: no wctting, wctting carly at nrst boll opcning, wctting
latc whcn most bolls wcrc opcn, and wctting both carly and latc. Plots wcrc all plantcd with P4498R.
Tc sccond cxpcrimcnt was a varicty trial in which cight rcplications ol lour varictics, Hammcr
(CPCS high yiclding, thincoatcd sccd varicty), P48R, ST998R and PHY470VR wcrc wcttcd
both carly and latc.
Ficld plots arc cstablishcd at Tc Univcrsity ol Arizona Maricopa Agricultural Ccntcr (MAC), and
a misting systcm lor incrcasing humidity in thc crop canopy was installcd. Vatcr was pumpcd lrom
thc irrigation ditch through two inch pipc throughout thc ncld to 20 20 lt plots. Tc misting
systcm consistcd ol !2 nvcgal/hr brass loggcrs spaccd about thrcc lcct apart in a !0 !0 lt grid ol
inch P\C pipc. 8ascd on 2004 humidity data, mistcrs wcrc raiscd to 2 lt abovc ground and misting
volumc incrcascd. Humidity and tcmpcraturc ol trcatcd plots was monitorcd using Hobo data loggcrs
to quantily impact ol wctting rcgimcn on canopy cnvironmcnt. Trcatmcnts consistcd ol two multiplc
day misting pcriods in AugustScptcmbcr (carly) and ctobcr (latc). Cotton was harvcstcd in carly
Novcmbcr and immcdiatcly ginncd.
Harvcstcd sccd will bc tcstcd lor aatoxin, and thc data uscd to continuc dcsign ol a ncld bascd
scrccning tcchniquc. Ultimatcly a modcl will bc dcvclopcd rclating wctting pcriod and tcmpcraturc to
aatoxin contcnt. Tis will contributc both to dcvclopmcnt ol thc scrccning tcchniquc and to a bcttcr
undcrstanding ol thc sccond phasc ol contamination. A modcl will bc usclul to growcrs in asscssing crop
aatoxin risk prior to harvcst and to rcscarchcrs in dcvclopmcnt ol altcrnativc aatoxin managcmcnt
stratcgics.
Corn Hybrids with Exotic Germplasm and Low-Afatoxin
Vcnwci Xu
!
,

Jinlcn Zhang
!
, Gary dvody
2
,

and V. Paul Villiams
1
!
2
Texas A&M University, Corpus Christi, TX;
1
USDA/ARS, Corn
Host Plant Resistance Research Unit, Mississippi State, MS
Aatoxin contamination ol corn is a chronic problcm in thc southcrn Unitcd Statcs. Vc obscrvcd that
drought tolcrant corn hybrids produccd a highcr yicld and had much lcss grain mold than susccptiblc
hybrids undcr drought conditions. Tc objcctivc ol this study was to dctcrminc il gcnctic improvcmcnt
ol abiotic strcss tolcrancc and corn carworm rcsistancc can rcducc thc aatoxin risk in this rcgion.
Tcn Tcxas xpcrimcntal Agricultural Station (TAS) cxpcrimcntal hybrids and nvc commcrcial chccks
(Pionccr hybrids 14K77 and 1!8!1, Garst 828, Triumph !4!6, and K XL269) wcrc grown in Lubbock,
Hallway, Corpus Christi, and 8ccvillc in Tcxas and Mississippi Statc, MS in 2001 and 2004. !n
Lubbock and Hallway, plants wcrc inoculatcd onc wcck altcr silking by injccting A. favus conidia into
silk channcls. !n Corpus Christi, 8ccvillc, and Mississippi Statc, corn kcrncls colonizcd by A. favus wcrc
distributcd bctwccn all rows whcn thc nrst hybrid was at thc midsilking stagc to providc thc incrcascd
and unilorm acrial disscmination ol conidia. !n all cascs, thc inocolum was lrom a high aatoxin
producing A. favus strain (NRRL117). A limitcd latc planting datc was uscd in Corpus Christi,
8ccvillc and Mississippi Statc to cncouragc scvcrc drought strcss at latcr stagcs ol maturity. Tc tcsts
uscd a randomizcd complctc block dcsign with ninc rcplications at Corpus Christi and 8ccvillc, lour
rcplications in Lubbock and Hallway, and thrcc rcplications in Mississippi. ars lrom cach plot wcrc
handharvcstcd. All cars wcrc thrcshcd and agronomic data wcrc rccordcd including grain yicld. Grain
samplcs wcrc ground in a Romcr mill and aatoxin 8 assay was donc on 0 g subsamplcs ol thc nncly
ground matcrial lor cach compositc rcplication using thc \icam immunoassay/uoromctcr systcm.
Twoycar rcsults showcd that S!V CML141 and S2871 NC100 had signincantly lowcr
aatoxin than thc control hybrids. Hybrid 8!!0 SGP1 had high yiclding and high aatoxin in most
cnvironmcnts in two ycars. !n 2001, thc aatoxin lcvcl in S!V CML141, S2871 NC100 and
P1!8!1 was 49, 1!, and !6! ppb rcspcctivcly at Corpus Christi, TX. Tc aatoxin ol thc Mississippi Statc
tcst in 2001 was gcncrally low and not signincant among thc cntrics. Tc rcsults in 2004 wcrc in gcncral
consistcnt with thc rcsults in 2001. !n 2004, thc aatoxin lcvcls in S!V CML141, S2871 NC100,
and P1!8!1 (CK) was rcspcctivcly .1, !6.7, and 70.0 ppb at Corpus Christi, !0.!, 9.4, .8 and 11.1 ppb
at Mississippi Statc. !n 2004, Lubbock had thc rainlall and tcmpcraturcs lavorablc lor corn growth and
dcvclopmcnt. Tc aatoxin lcvcls undcr wcllwatcrcd and drought strcsscd tcst in Lubbock wcrc similar,
although thc avcragc grain yicld ol thc !4 cntrics dcclincd lrom !84 bu/a in wcllwatcrcd condition to
!26 bu/a in drought strcsscd condition. Tc aatoxin lcvcls in S!V CML141, S2871 NC100,
and P1!8!1 was 90.0, 10., and 260.0 ppb undcr wcllirrigatcd conditions (mcan ol !4 hybrids as !14.8
ppb), whilc undcr drought conditions wcrc 91., 11., and 240 ppb (mcan ol !4 hybrids as !20. ppb).
Tcsc low aatoxin hybrids yicldcd wcll in comparison to thc chccks. For cxamplc, thc avcragc yicld
ol S!V CML141 in Hallway and Lubbock in 2001 and 2004 was 228 and 220 bu/a whilc P1!8!1
produccd 222 and 24 bu/a. Tc S!V CML141 is a whitc hybrid. !n othcr ncld trials in south Tcxas
in 200, this hybrid produccd !!9 and !7 bu/a at Vcslaco and Ganado in comparison to !!! and !18
bu/a ol P1!8!1. Tcsc rcsults indicatc that brccding lor drought tolcrancc and carworm rcsistancc is a
promising approach to rcducc aatoxin contamination in corn grown in Southcrn cnvironmcnts. Somc
ol our cxpcrimcntal hybrids havc comparablc yicld yct signincantly low aatoxin in comparison to thc
commcrcial hybrids. Tc TAS cxpcrimcntal hybrids and thcir parcntal lincs havc at lcast 2 tropical
gcrmplasm and wcrc sclcctcd lor drought and hcat tolcrancc, CV rcsistancc and ovcrall agronomic
pcrlormancc. Tcy havc tight husk, good car tip covcragc, signincantly lowcr grain mold and lcss car
injurics by corn carworm.
Computational Tools for Protein Identifcation and Gene Ontology Annotation
of the Maize Proteome
Susan M. 8ridgcs
!
, Julia . Hodgcs
!
, Grcgory 8rycc Magcc
!
, Nan Vang
!
, awn S. Luthc
2
, and
V. Paul Villiams
1
!
Department of Computer Science and Engineering, Mississippi State University, Mississippi State, MS;
2
Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, MS;
1
USDA ARS Corn Host Plant Resistance Research Unit, Mississippi State, MS
Tc Corn Host Plant Rcsistancc Rcscarch Unit, USAARS, Mississippi Statc Univcrsity in
collaboration with r. awn Luthc, is conducting protcomics studics to dctcrminc thc cccts ol biotic
and abiotic lactors on Aspergillus favus inlcction and aatoxin accumulation in maizc. Computational
support has providcd scicntists with tools to improvc protcin idcntincation ratcs and providc cmcicnt
and inlormativc annotation ol thc protcomc.
Charactcrization ol thc maizc protcomc ol thc dcvcloping car undcr dicrcnt conditions has thc
potcntial to rcvcal thc lundamcntal proccsscs that conlcr rcsistancc in somc ccll lincs. Advanccs in
protcomics havc bccn madc possiblc by highthroughput mcthods lor gcl clcctrophorcsis and ncw
tcchnologics lor mass spcctromctry such as LC/MS/MS. Vc havc prcviously rcportcd thc dcvclopmcnt
ol thc P! databasc ol translatcd STs and havc shown that usc ol this databasc lor protcin
idcntincation lor a 2dimcnsional gcl cxpcrimcnt with cob protcins rcsults in idcntincation ol 87. ol
thc spots comparcd to a 6 idcntincation ratc with thc NC8! databasc nonrcdundant grccn plant
databasc. Tc P! databasc pipclinc has bccn parallclizcd and now runs on a high pcrlormancc clustcr,
making it possiblc to rapidly gcncratc updatcd databascs.
Additional tools havc bccn dcvclopcd to strcamlinc thc protcin idcntincation proccss and to providc
thc Gcnc ntology annotation ol thc idcntincd protcins. Tc multidimcnsional protcin idcntincation
tcchnology (MudP!T) can bc uscd to scparatc many hundrcds to thousands ol pcptidcs in a singlc
cxpcrimcnt. Tc rcsults obtaincd lrom Scqucst analysis ol MudP!T cxpcrimcnts can bc quitc challcnging
to analyzc, particularly whcn thc databasc uscd lor qucrics is highly rcdundant. Tis is thc casc whcn
using translatcd STs bccausc many corrcspond to thc samc protcin or to closcly rclatcd protcins.
Scicntists typically must intcgratc inlormation lrom scvcral rcpctitions and data sourccs to dctcrminc
conndcncc in an idcntication. Tc PcpSort tool was dcvclopcd to assist with this typc ol analysis. Tc
tool combincs multiplc rcps sclccting thc bcst scorc lor cach pcptidc bascd on a uscr spccincd scoring
systcm. Potcntial protcin duplicatc idcntincations arc collcctcd and prcscntcd to thc uscr simultancously
so thc uscr can sclcct thc bcst idcntincation and climinatc duplicatcs. Scorcs and counts lor pcptidcs arc
updatcd automatically whcn duplicatcs arc rcmovcd.
Vc havc dcploycd thc MaizcG databasc, as part ol Agbasc www.agbasc.msstatc.cdu, a curatcd,
opcnsourcc, Vcbacccssiblc rcsourcc lor lunctional analysis ol agricultural plant and animal gcnc
products. Four tools havc bccn dcvclopcd as part ol Ag8asc to support Gcnc ntology annotation ol
protcins lrom high throughput cxpcrimcnts: G Rctricvcr, G annotator, G Pronlcr and G Slim
\icwcr. G Rctricvcr providcs a nrst pass rctricval ol G annotations that arc currcntly publishcd
lor rcsourccs acccsscd by thc Ag8asc databasc. G Annotator is uscd to supplcmcnt thc annotations
providcd by G Rctricvcr by providing prcdictions ol G annotations lor protcins bascd on homology
with annotatcd protcins bascd on uscrsclcctcd 8LAST paramctcrs. G Pronlcr providcs an ovcrvicw
ol G associations availablc lor a uscrspccincd spccics including thc numbcr ol G associations and
thc numbcr ol annotatcd protcins. Tc G Slim \icwcr providcs an ovcrvicw ol thc mcmbcrship in
G catcgorics ol a protcin data sct using catcgorics dcnncd in a G Slim. utput is in a lorm that can
bc casily importcd into xccl lor lormatting as a pic chart.
Progress in Breeding Peanut for Resistance to Preharvest Afatoxin
Contamination and Drought
C.C. Holbrook
!
, 8.Z. Guo
!
, P. Timpcr
!
, .M. Vilson
2
, . Sullivan
!
, . Cantonwinc
2
, and
C. Kvicn
2
!
2
University of Georgia, Tifton, GA
Aatoxin contamination costs thc U.S. pcanut industry ovcr 820 million annually. Tc dcvclopmcnt
ol pcanut cultivars with rcsistancc to prcharvcst aatoxin contamination (PAC) would rcducc thcsc
costs. Vc havc dcvclopcd ncld scrccning tcchniqucs that can mcasurc gcnctic dicrcnccs in aatoxin
contamination, and havc uscd thcsc tcchniqucs to idcntincd !! corc acccssions that havc shown at lcast
a 70 rcduction in PAC in multiplc cnvironmcnts. Vc havc also idcntincd signincant rcduction in PAC
in pcanut gcnotypcs with drought tolcrancc. Tcsc sourccs ol rcsistancc to PAC havc bccn cntcrcd into
a hybridization program. Tcy havc bccn crosscd with cultivars and brccding lincs that havc high yicld,
acccptablc gradc, and rcsistancc to tomato spottcd wilt virus (TSV\). uc to thc largc cnvironmcntal
variation in PAC, it is not lcasiblc to cxaminc thcsc brccding populations until latc gcncrations whcn
thcrc is lcss hctcrozygosity and adcquatc sccd arc availablc lor ncld tcsting using multiplc rcplications.
Vc havc cxamincd numcrous brccding populations and havc idcntincd scvcral lamilics and individual
brccding lincs that havc rclativcly low PAC, rclativcly high yicld, and acccptablc lcvcls ol rcsistancc to
TSV\. Howcvcr, much lastcr brccding progrcss could bc achicvcd through thc dcvclopmcnt and usc
ol indircct sclcction tcchniqucs. Vc arc cxploring this with studics on mcchanisms ol rcsistancc to
PAC and attcmpting to dcvclop molccular markcrs lor rcsistancc. Tc most promising mcchanisms
wc havc idcntincd thus lar arc rcsistancc to drought and rcsistancc to thc pcanut rootknot ncmatodc.
Vc havc dcvclopcd scvcral latc gcncration brccding lincs with rcsistancc to drought. Tcsc lincs havc
cxhibitcd rcduccd aatoxin contamination in multiplc cnvironmcnts. Rcccntly, wc havc dcvclopcd latc
gcncration brccding linc with rcsistancc to TSV\ and thc pcanut rootknot ncmatodc that appcar to
havc agronomically acccptablc yicld and gradc. Tcsting is ongoing to dctcrminc il thcsc lincs will havc
rcduccd aatoxin contamination.
Searching for New Resistance and Control Measures of Afatoxin in Corn
Stcvcn Moorc
!
, Hamcd Abbas
2
, and Mark Millard
1
!
Louisiana Agricultural Experiment Station Louisiana State University Agricultural Center, Alexandria,
LA;
2
USDA-ARS, Crop Genetics and Production Research Unit, Stoneville, MS;
1
North Central Regional
Plant Introduction Station Iowa State University, Ames, IA
Tc mission ol aatoxin rcscarch in thc LSU AgCcntcr is to rcducc or climinatc aatoxin contamination
in corn. Spccinc objcctivcs includc idcntilying ncw gcrmplasm with improvcd rcsistancc, using glulosinatc
to rcducc aatoxin contamination, and cvaluating thc usc ol atoxigcnic Aspergillus favus strains. !n thc
200 Southcastcrn Rcgional Aatoxin Tcst (SRAT) in Ccntral Louisiana, numcrous hybrids lrom
multiplc brccding programs wcrc idcntincd that had highcr yiclds and lowcr aatoxin than commcrcial
chccks, indicating that progrcss is bcing madc in dcvcloping commcrcially viablc cultivars. Tx 807
(P!6!9410), TZ!!8 (P!0621), Haiti 11 (P!481902), AR!P69 (P!2!8!89) and CML 41 (P!91) had
lowcr aatoxin than thc Mp1!1871 and Tcx6871 rcsistant chccks in thc 200 scrccning trial. A corn
linc lrom Sundancc Gcnctics had thc lowcst aatoxin contamination in matcrial analyzcd lrom thc
200 scrccning trial up to now. Glulosinatc trcatmcnt lowcrcd aatoxin contamination in thc non
Libcrty Link corn hybrid N81Z8 but sccmcd to havc littlc or no ccct in Libcrty Link corn hybrid
N81N. Glulosinatc lowcrcd aatoxin morc whcn applicd sooncr altcr midsilk than whcn applicd
latcr altcr midsilk. Tc avcragc aatoxin contamination in corn spraycd with 4.2 and 8. ounccs
ol glulosinatc wcrc signincantly lowcr than thc control and rcduccd aatoxin by about 4 in car
inoculatcd trcatmcnts and by about 86 in groundinoculatcd trcatmcnts. Glulosinatc application to
corn thrcatcncd by aatoxin contamination appcars to bc a hopclul cconomic tool lor produccrs. Morc
ncldplot and ncldscalc rcscarch is nccdcd to connrm bcncnts and dctcrminc bcst practiccs. Tcrc
appcarcd to bc a small but bcncncial ccct ol applying thc atoxigcnic strain K49 on rcducing aatoxin
in corn. Atoxigcnic strains may bc a usclul tool in rcducing aatoxin but much work rcmains to nnd thc
most ccctivc strains and to dctcrminc ncld application stratcgics.
Development of Afatoxin-resistant Maize Inbreds and Identifcation of
Potential Resistance Markers through USA-Africa Collaborative Research
Robcrt L. 8rown
!
, ZhiYuan Chcn
2
, Abcbc Mcnkir
1
, and Tomas . Clcvcland
!
!
Southern Regional Research Center, USDA-ARS, New Orleans, LA;
2
Department of Plant Pathology
and Crop Physiology, Louisiana State University, Baton Rouge, LA;
1
International Institute of Tropical
Agriculture, Ibadan, Nigeria
A rcscarch collaboration bctwccn thc Southcrn Rcgional Rcscarch Ccntcr (SRRC) and thc !ntcrnational
!nstitutc ol Tropical Agriculturc (!!TA) was initiatcd in !998. Tc purposc ol this collaboration is to
dcvclop aatoxinrcsistant maizc inbrcds lor usc in Vcst and Ccntral Alrica and lor usc in thc U.S. as
wcll. Anothcr objcctivc is to idcntily markcrs in thc maizc lincs gcncratcd through this collaboration to
lacilitatc markcrassistcd translcr ol rcsistancc traits. 1 S tcmpcratc lincs and 9! S tropical lincs havc
bccn advanccd to S and S rcspcctivcly. 44 S lincs showing good agronomic traits wcrc scnt to thc U.S.
lor analysis using thc kcrncl scrccning assay (KSA). 44 additional ncw inbrcds lrom dicrcnt crosscs
wcrc scrccncd in thc U.S., thus lar 6 show lcvcls as low or lowcr than rcsistant inbrcd, M!82. Most
advanccd inbrcds with dcsirablc agronomic charactcristics wcrc plantcd in thc dry scason to gcncratc
hybrids to tcst lor agronomic pcrlormancc in at lcast 2 locations in 200. Ncw inbrcd lincs lrom this
program, oncc rcsistancc and agronomic traits arc connrmcd, will bc rclcascd as sourccs ol gcncs lor
rcsistancc to U.S. brccding programs and will bc uscd lor dcvclopmcnt ol hybrids and synthctics in
Alrican national programs. 8asic gcnctic inlormation will also bc gcncratcd using ncar isogcnic lincs
with contrasting lcvcls ol aatoxin to dcvclop a brccding stratcgy lor pyramiding dicrcnt allclcs that
conlcr rcsistancc to mycotoxins. Protcomc analysis ol pairs ol closclyrclatcd S lincs havc dcmonstratcd,
as in a prcvious study cmploying dicrcnt gcrmplasm, constitutivclycxprcsscd strcssrclatcd protcins
that arc associatcd with rcsistancc. A prcviously undcscribcd bcta!, 1glucanasc, also associatcd with
rcsistancc, was idcntincd in this invcstigation and cloncd. Glyoxalasc ! and PR !0 protcins, prcviously
idcntincd through protcomics as associatcd with rcsistancc, arc bcing invcstigatcd in RNAi gcnc
silcncing studics. Sccd has bccn produccd lrom RNAi translormations and is bcing charactcrizcd lor
gcnc cxprcssion lcvcls and lor aatoxin accumulation. Ncw RNAi studics will locus on trypsin inhibitor
and scrinc/thrconinc kinasc.
PANEL DISCUSSION: Crop Resistance Conventional Breeding
Panel Chair: on Vhitc
Panel Members: Javicr 8ctrn, Kcrry Mayncld, avid Kcndra, Tom Gradzicl, 8aozhu Guo,
Tom 8rooks, awn Luthc, Mary lson, Vcnwci Xu, Susan 8ridgcs, Corlcy Holbrook, Stcvc
Moorc, Paul Villiams, and 8ob 8rown
Summary of Presentations: Prcscntations includcd in thc convcntional brccding scssion varicd grcatly
in approach and goals. Scvcral projccts arc conccntrating on idcntincation ol ncw sourccs ol rcsistancc
mostly lrom cxotic gcrmplasm. thcr projccts arc combining idcntincation ol sourccs ol rcsistancc with
applicd brccding programs that arc conccntrating on low aatoxin as wcll as agronomic charactcristics
that will bc rcquircd bclorc rcsistant varictics will bc uscd by larmcrs. Somc arc using molccular markcr
assistcd back crossing to movc chromosomc rcgions associatcd with rcsistancc lrom agronomically
poor inbrcds into commcrcially acccptablc inbrcds. thcr projccts arc using gcnomic and protcomics
approachcs in an attcmpt to bcttcr undcrstand thc naturc ol rcsistancc.
Trcmcndous progrcss has bccn madc in thc brccding ol rcsistant pcanuts. Vith pcanuts it is ncccssary
to havc gradc, virus rcsistancc and othcr traits bclorc varictics can bc commcrcially uscd. Vith corn, a
numbcr ol projccts arc dcvcloping hybrids with cmphasis on rcsistancc to aatoxin production couplcd
with yicld, rcsistancc to root and stalk lodging, and othcr agronomic charactcristics. Tc projccts on
corn havc joincd lorccs to cvaluatc hybrids produccd in cvcryoncs projcct and comparc thcm with
commcrcially uscd corn hybrids. Vith trcc nuts rcscarch has conccntratcd on avoidancc ol inscct damagc
which is highly corrclatcd with pcnctration ol Aspcrgillus spccics and thc production ol aatoxin.
Summary of Panel Discussion: Tcrc was discussion on how wc can utilizc data dcvclopcd with gcnomics
and protcomics in applicd brccding programs. Tc pancl agrccd that it will takc somc timc bclorc
gcnomics approachcs can bc dircctcd toward applicd brccding programs. !t was suggcstcd that thc
opportunity cxists with corn to look at gcnomics and protcomics data associatcd with chromosomc
rcgions whcrc molccular markcrs havc shown to bc associatcd with gcncs lor rcsistancc. Tat would
cnhancc thc possibility ol nnding spccinc gcncs, cspccially lrom thc rcsistant inbrcd linc Mp1!1.
Tcrc was also discussion ol cxactly how Hazard Analysis Critical Control Point (HACCP) rclatcs to
rcsistancc. !t was pointcd out that HACCP is actually a highly structurcd and systcmatic way to addrcss
important brccding goals. Also, thcrc was discussion on thc usc ol Libcrty hcrbicidc on nonLibcrty
Link corn hybrids to rcducc aatoxin. 8asically thc hcrbicidc Libcrty is applicd altcr pollination on
hybrids susccptiblc to thc hcrbicidc. Tis is bccn shown to rcducc aatoxin. !t was suggcstcd that thc
modc ol action ol Libcrty hcrbicidc is production ol ammonia which is known to rcducc aatoxin.
Tcrc was gcncral agrccmcnt among thc pancl that outstanding progrcss has bccn madc with rcspcct
to rcsistancc to aatoxin production.
Multilocation Evaluation of Afatoxin Accumulation in Yellow Maize Hybrids
Cody McKcc, Tom !sakcit, Gary dvody, Kcrry Mayncld, Javicr 8ctrn
A major obstaclc in maizc production across thc Southcrn U.S. and othcr parts ol thc world is
accumulation ol aatoxin, a known carcinogcn in both humans and livcstock produccd by Aspergillus
favus. Aatoxin contamination is dimcult to cvaluatc in thc ncld bccausc ol varying amounts ol
sourcc inoculum and dcpcndcncy on lavorablc cnvironmcntal conditions. Tcxas is an cxccllcnt arca lor
cxamining aatoxin accumulation bccausc ol thc tcndcncics lor abiotic strcsscs such as drought and
high tcmpcraturcs. ur objcctivcs wcrc to !) cstimatc thc rcsponscs ol thcsc hybrids to aatoxin across
a rangc ol cnvironmcnts, 2) idcntily thc hybrids within cach group that cxhibitcd thc lowcst lcvcls ol
contamination, 1) analyzc thc rclationship bctwccn agronomic pcrlormancc and aatoxin accumulation,
and 4) dctcrminc how much gcnotypc cnvironmcnt intcraction (G!) acct thcsc traits. !n thc past,
our program has cxamincd aatoxin accumulation at thrcc cnvironmcnts in Southcrn Tcxas, Vcslaco,
Corpus Christi and Collcgc Station. Conccrn has bccn raiscd that this is not sumcicnt to cxaminc
gcnotypc cnvironmcntal cccts. Tcrclorc, during 200 twcnty nvc hybrids, 20 cxpcrimcntal tcstcrosscs
with inbrcds LH!9 and LH2!0 and commcrcial hybrids (P1!8!1, P12R2, 8H89!1, KC6972 and
V4700), wcrc cvaluatcd undcr inoculation with A. favus in cight locations rcprcscnting thc maizc
producing rcgions ol Tcxas. Aatoxin conccntration was 407 ng g
' at Collcgc Station, 86 ng g
' at
Vcslaco, !094 ng g
' at Corpus Christi, !1! ng g
' at Castrovillc, 1! ng g
' at Vharton, 407 ng g
'
at Grangcr, 209 ng g
' at 8ardwcll, and 274 ng g
' at Prospcr. vcrall, wc lound that thc commcrcial

hybrids had highcr grain yiclds than thc cxpcrimcntal hybrids, with P1!8!1 yiclding thc highcst at 6.4
Mg ha
'. Howcvcr, cxpcrimcntal hybrids, cspccially tcstcrosscs with LH!9, wcrc lcss susccptiblc to
aatoxin accumulation than commcrcial hybrids. Hybrid TxLAMA200242/LH!9 had thc lowcst
avcragc aatoxin accumulation across locations at !4 ng g
'. Aatoxin conccntration was positivcly

corrclatcd with ol car rot and tcst wcights and ncgativcly corrclatcd with grain yicld and !000 kcrncl
wcight. Vc obscrvcd a signincant G! lor both aatoxin conccntration and grain yicld. Tcrclorc,
multiplc locations arc ncccssary lor cstimating agronomic pcrlormancc and rcsponsc to aatoxin ol
maizc hybrids.
Southern East Regional Afatoxin Test (SERAT)
Michacl Clcmcnts
!
, Paul Villiams
!
, Stcvc Moorc
2
, Matthcw Krakowsky
1
, 8aozhu Guo
1
, on
Vhitc
4
, Vcnwci Xu
, Tom !sakcit
6
, Tom 8rooks
!
, Gary Vindham
!
, Hamcd Abbas
7
, Jamcs
Pcrkins
8
, anicl Gorman
9
, Quinton Raab
!0
, Kcith Arnold
!0
, avid Smith
!!
, and Javicr 8ctrn
6
!
2
Louisiana State University, Alexandria, LA;
1
USDA-ARS, Tifton,
GA;
4
University of Illinois, Urbana, IL;

6
Texas A&M University,
7
USDA-ARS, Stoneville, MS;
8
Monsanto Company Crop Protection, Waterman, IL;
9
Pioneer Dupont, Cairo, GA;
!0
BH Genetics, Moulton, TX;
!!
Zea Sage, Sycamore, IL
Aatoxin contamination ol corn grain is a chronic problcm lor growcrs in thc southcast Unitcd Statcs.
For scvcral ycars, rcscarch groups at Louisiana, Mississippi, Gcorgia, !llinois and Tcxas havc bccn
scrccning corn gcrmplasm lor rcsponsc to aatoxin contamination at spccinc locations. Although
scvcral sourccs ol rcsistancc havc bccn idcntincd and rclcascd, at prcscnt, thcrc arc no clitc inbrcd lincs
rcsistant to aatoxin that can bc uscd dircctly in commcrcial hybrids. Aatoxin accumulation is scvcrcly
acctcd by thc cnvironmcnt. Gcnotypc by cnvironmcnt intcraction (G!) is normally signincant with
gcnotypcs showing dicrcnt rclativc rcsponsc across cnvironmcnts. A tcsting nctwork ol cnvironmcnts
across major growing arcas acctcd by aatoxin has bccn cstablishcd to idcntily thc most consistcnt
stablc sourccs ol rcsistancc. SRAT is a multilocation and multistatc rcgional tcst ol thc most
promising gcrmplasm lrom cach brccding program. Participants providc sccd ol a lcw hybrids and a
tcsting location. valuations arc conductcd undcr inoculation with A. favus lollowing thc protocols
commonly uscd by cach rcscarch group. !n addition to aatoxin, grain yicld and othcr agronomic traits
such as maturity, lodging, grain moisturc, tcst wcights, ctc. arc rccordcd. !n 2004, SRAT tcsts wcrc
conductcd at six locations: Alcxandria, LA, Tilton, GA, Starkvillc, MS, Urbana, !L, Hallway, TX,
and Vcslaco, TX. Tc silk channcl inoculation mcthod was uscd at all locations cxccpt Urbana and
Alcxandria, whcrc inoculation with a pinboard was uscd, and Starkvillc, whcrc inoculum was injcctcd
through husk lcavcs into thc sidc ol thc car. Aatoxin conccntration was variablc across locations.
Avcragc aatoxin was 710 ng g
' at Alcxandria, 4 ng g
' at Tilton, 192 ng g
' at Starkvillc, !82 ng

g
' at Hallway, 2! ng g
' at Urbana and 62 ng g
' at Vcslaco. Signincant G! was obscrvcd lor both

aatoxin and grain yicld. Principal componcnt analysis ol aatoxin conccntrations suggcstcd dicrcnt
rcsponsc ol hybrids to thc dicrcnt locations. !n 200, SRAT tcsts wcrc conductcd at 9 locations:
Alcxandria, LA, Tilton, GA, Starkvillc, MS, Urbana, !L, Hallway, TX, and Vcslaco, TX, Ganado, TX,
Mistic, GA, and Claxton, G. Similar inoculations tcchniqucs as in 2004 wcrc uscd. Tcsts at Mistic
and Claxton, GA wcrc not inoculatcd. Avcragc aatoxin was 27 ng g
' at Alcxandria, 87 ng g
' at
Tilton, !!8 ng g
' at Ganado, and !14 ng g
' at Vcslaco. Aatoxin conccntrations lor thc rcst ol thc

locations arc bcing quantincd. Grain yicld was also variablc across locations. Avcragc grain yicld was 88
bu/a at Vcslaco, !66 bu/a at Mistic and Claxton combincd, !!7 bu/a at Ganado, !1! bu/a at Tilton, !29
bu/a at Starkvillc, !!8 bu/a at Hallway, and !!6 bu/a at Alcxandria. As in 2004, highly signincant G!
was obscrvcd lor grain yicld with cnvironmcnts discriminating dicrcntly tcsting hybrids. Rcsponsc ol
matcrials lrom dicrcnt programs was variablc, in that hybrids showcd dcsirablc cxprcssion lor dicrcnt
traits such as aatoxin, grain yicld and standability. Tis suggcsts possibilitics ol combining positivc
traits by crossing gcrmplasm lrom dicrcnt programs. !n gcncral, cxpcrimcntal hybrids havc shown
lcss susccptibility to aatoxin but lcss grain yicld than commcrcial chccks. Locations discriminatcd
corn hybrids dicrcntly lor both aatoxin contcnt and grain yicld. SRAT has promotcd collaboration
and idcntincd complcmcntary gcrmplasm lrom dicrcnt programs. Vith this collaborativc rcgional
tcsting, wc cxpcct to idcntily thc most stablc sourccs ol aatoxin rcsistancc, asscss thcir consistcncy
across dicrcnt cnvironmcnts and trcatmcnts, charactcrizc thcir agronomic pcrlormancc, incrcasc thc
collaboration among rcscarch groups in dicrcnt statcs, and to asscss thc magnitudc and naturc ol
gcnotypc cnvironmcnt intcraction lor aatoxin.
Evaluation of CIMMYT Germplasm for Response to Afatoxin Production in
the Southern USA
an Jccrs
!
, Matt Krakowsky
2
, Paul Villiams
1
, Javicr 8ctrn
4
!
CIMMYT, Mexico D.F.;

1
Texas A&M
University, College Station, TX
Tc dcmand lor salc, nutritious corn rcquircs corts to dcvclop improvcd hybrids with bcttcr lood
proccssing and nutritional qualitics. xotic whitc and ycllow lincs rcprcscnt a sourcc ol gcncs lor
quality traits. Tc !ntcrnational Maizc and Vhcat !mprovcmcnt Ccntcr (C!MMYT), has dcvclopcd
gcrmplasm morc tolcrant to abiotic strcsscs and rcsistant to biotic strcsscs, lor targcting aatoxin pronc
arcas cspccially in Alrica. ur objcctivc was cvaluatc thc rcsponsc ol sclcctcd C!MMYT whitc and
ycllow corn inbrcds and hybrids to aatoxin contamination in Southcrn USA, and to dctcrminc thc
gcnctic variability that cxists lor rcsistancc to Aspergillus car rot in C!MMYT gcrmplasm lor usc by
US invcstigators, C!MMYT, and thcir nctwork ol partncrs in dcvcloping countrics who nccd maizc
varictics with improvcd grain quality and storability. Twcnty nvc C!MMYT ycllow hybrids, twcnty
nvc whitc hybrids, twcnty lour whitc inbrcds and twcnty cight ycllow inbrcds wcrc or arc currcntly
bcing cvaluatcd in Tcxas, Mississippi and Gcorgia. Tcsc inbrcds and hybrids wcrc sclcctcd bascd on
low lcvcls ol Aspergillus favus car rot inlcction undcr ncld cvaluations using artincial inoculations in
Mcxico. U.S. cvaluations in 200, wcrc artincially inoculatcd with Aspergillus favus isolatc NRRL117
two wccks altcr owcring using thc silk channcl inoculation mcthod in Starkvillc, MS and thc
colonizcd kcrncl mcthod in Vcslaco, TX. Quantincation ol aatoxin was conductcd using thc \icam
Aatcst (Vatcrtown, MA). Tcrc wcrc signincant dicrcnccs lor aatoxin contcnt in both inbrcds and
hybrids. Vhitc and ycllow inbrcds wcrc cvaluatcd in Starkvillc, MS. Somc gcnotypcs did not owcr
carly cnough lor inoculations, and no aatoxin data was collcctcd. Avcragc aatoxin conccntration was
608 ng g
' lor whitc inbrcds and 4! ng g
'

lor ycllow inbrcds. Vhitc quality protcin maizc (QPM)
inbrcd CML!42 and ycllow QPM inbrcd CLQG207 had aatoxin lcvcls bclow thc rcsistant chccks,
Mp1!1 and Mp7!7, rcspcctivcly. Avcragc aatoxin conccntrations lor whitc hybrids wcrc !48 ng g
'

in Starkvillc, MS and !7 ng g
'

in Vcslaco, TX. nvironmcntal conditions wcrc not conducivc lor
Aspergillus inlcction and aatoxin production with thc colonizcd kcrncl inoculation tcchniquc uscd
in Vcslaco. Scvcral whitc hybrids (c.g., CML14! CML24, CML14! CML49) had lowcr
aatoxin than thc most rcsistant chcck. Avcragc aatoxin conccntrations lor ycllow hybrids wcrc 81 ng
g
'

in Starkvillc, MS and 18 ng g
' in Vcslaco, TX. Ycllow hybrids CML4! CL02844 and CL

0240 CML44 had lowcr aatoxin lcvcls than rcsistant chccks in Starkvillc, MS. Lcss variation lor
aatoxin lcvcls was obscrvcd in Vcslaco, TX. vcrall, signincant dicrcnccs lor aatoxin conccntration
wcrc obscrvcd lor both C!MMYT whitc and ycllow maizc inbrcds and hybrids. Gcnctic variation, bascd
on dicrcnccs in thc production ol aatoxins, was obscrvcd among cxotic C!MMYT gcrmplasm, whcrc
somc C!MMYT inbrcds and hybrids had aatoxin conccntrations similar to thc most rcsistant chccks.
Multilocation and multiycar cvaluations would bc nccdcd to sclcct thc most promising gcrmplasm to
introgrcss in U.S. brccding programs.
Phenotypic and Genotypic Characterization of a RIL Maize Mapping
Population for Afatoxin and Secondary Traits
Mclanic dwards, Monica Mcnz, Tom !sakcit and Javicr 8ctrn
Aatoxin is a mycotoxin produccd by Aspergillus favus that is toxic to both humans and livcstock.
8rccding corts to producc commcrcial hybrids rcsistant to aatoxin would bc cnhanccd by bcttcr
undcrstanding ol thc gcnctic componcnts involvcd. A rccombinant inbrcd linc (R!L) population was
dcvclopcd using phcnotypically divcrgcnt parcntal inbrcds CML!76 and Tx8!! to map quantitativc trait
loci (QTL) lor rcsponsc to aatoxin and root lodging. Tis population ol !62 S6 R!Ls, was cvaluatcd
in rcplicatcd trials in two Tcxas locations, Vcslaco and Collcgc Station undcr inoculation. Root
lodging, aatoxin conccntration, maturity, cndospcrm tcxturc, and kcrncl intcgrity wcrc all mcasurcd
lor thc population. Hcritability lor cach trait, and phcnotypic and gcnotypic corrclations ol sccondary
charactcristics to aatoxin, wcrc cstimatcd lrom variancc componcnts. Tc mapping population showcd
signincant dicrcnccs and broad rangcs lor all agronomic traits studicd, with thc ospring showing
transgrcssivc scgrcgation lor cach ol thc traits. Tc trials had scvcrc root lodging as a conscqucncc ol
hcavy winds bclorc or around owcring timc. Lincs had signincant dicrcnccs lor root lodging with
CML!76 bcing morc susccptiblc than Tx8!!. Mcan aatoxin conccntration was highcr at Collcgc
Station (292 ng g
') than Vcslaco (2!4 ng g
'). vcrall mcan lor thc population across locations was

20 ng g
'. Tc population mcan lor kcrncl intcgrity was 2.6 (in a visual scalc lrom ! to ), which was
closcr to thc avcragc lor Tx8!! (2.8) than that ol CML!76 (!.7). Tc population mcan lor cndospcrm
tcxturc was 2.2 (in a visual scalc lrom ! to ), intcrmcdiatc bctwccn thc mcans lor parcntal lincs Tx8!!
(2.7) and CML!76 (!.6). Kcrncl !ntcgrity was thc trait that was most highly gcnotypically corrclatcd
to aatoxin conccntration (0.847 at Collcgc Station, 0.70 at Vcslaco). CML!76 has a lowcr kcrncl
intcgrity rating (thus morc intact kcrncls) than Tx8!!, as wcll as having a lowcr aatoxin conccntration.
ndospcrm tcxturc was also highly corrclatcd to aatoxin conccntration (0.46 at Collcgc Station,
0.492 at Vcslaco), although lcss so than kcrncl intcgrity. Tc inty cndospcrm ol CML!76 is corrclatcd
to a lowcr aatoxin conccntration than oury cndospcrm likc that ol Tx8!!. Hcritability cstimatcs at
Vcslaco wcrc high lor all studicd traits. Hcritability lor aatoxin conccntration was 0.12 at Collcgc
Station and 0.66 at Vcslaco. Tc two traits corrclatcd to aatoxin accumulation, cndospcrm tcxturc and
kcrncl intcgrity, wcrc both highly hcritablc (0.8 and 0.76, rcspcctivcly) and also casy to sclcct lor in
thc ncld. Tis providcs possibilitics lor luturc sclcction indiccs that may cxpcditc sclcction lor aatoxin
rcsistancc and providc a morc incxpcnsivc initial sclcction critcrion. Tc population was thcn gcnotypcd
using simplc scqucncc rcpcat (SSR) markcrs, and markcr data comparcd to phcnotypic data to asccrtain
associations bctwccn loci and rcsponsc to aatoxin or root lodging by using singlc markcr analysis in
SAS. !n prcliminary analysis, scvcral markcrs wcrc signincantly associatcd with root lodging per se
(umc2!61, umc2!80, umc!!24) and with rcsponsc to aatoxin (hpi072, phi087). Additional gcnotyping
and markcr analysis is undcrgoing.
Expression of LOX Pathway Genes in Corn Embryos Associated with Aspergillus
favus Resistance
Albcrto Camas
!
, L. Lopcz
!
, P. Villiams
2
and .S. Luthc
!
!
Department of Biochemistry and Molecular Biology, Mississippi State University, MS;
2
USDA-ARS, Corn
Host Plant Resistance Research Unit, Mississippi State University, MS
Tc accumulation ol aatoxin, a mycotoxin produccd by thc lungus Aspergillus favus during maizc
grain nll continucs to do bc a problcm. 8ccausc most aatoxin problcms dcvclop in thc ncld. Tc bcst
stratcgy lor climinating mycotoxin production is to dcvclop prcharvcst host rcsistancc to aatoxin
contamination. USAARS scicntists at Mississippi Statc Univcrsity havc contributcd to this intcnsc
ncld rcscarch by rclcasing scvcral corn inbrcds as a sourcc ol rcsistancc to kcrncl inlcction by A. favus.
Howcvcr, incorporating rcsistancc lrom thcsc sourccs into commcrcial hybrids rquircs idcntincation
and charactcrization ol lactors shown to bc associatcd with rcsistancc. Tc ability to idcntily molccular
markcrs associatcd with rcsistancc would hclp to advancc thc brccding program and providc clucs about
thc mcchanisms ol rcsistancc. Vc lound scvcral lox pathway gcncs associatcd with corn rcsistancc. 8y
using QTPCR wc comparcd lox, aos, and opr cxprcssion lcvcls ol maturc and immaturc cmbryos lrom
corn inbrcds and dicrcnt hybrids bctwccn rcsistant and susccptiblc inbrcd gcnotypcs. Tc dicrcnccs
in gcnc cxprcssion bctwccn rcsistant and susccptiblc gcnotypcs could bc rclatcd to thc plant rcsistancc
mcchanisms. Tcrclorc wc proposc somc ol thc LX pathway gcncs as potcntial molccular markcrs
that could contributc to gct commcrcially availablc and agronomically acccptablc corn lincs.
Breeding for Increased Resistance to Fusarium verticillioides in Maize
Magcn Starr
!
, Lcilani Robcrtson
!, 2
, Jamcs Holland
!, 1
and Gary Paync
2
!
Department of Crop Science, North Carolina State University, Raleigh, NC;
2
Department of Plant Pathology,
1
USDA, ARS, Plant Science Research Unit, North Carolina
State University, Raleigh, NC
Fusarium car rot in maizc is most oltcn thc rcsult ol colonization by Fusarium verticillioides
(lormcrly F. moniliforme) which produccs lumonisin, a mycotoxin rcsponsiblc lor discascs such as
lcukocnccphalomalacia (LM) in horscs, and pulmonary cdcma (PS) in hogs, and is corrclatcd with
csophagcal canccr in humans.
Prcvious studics in maizc havc dcmonstratcd that thcrc is gcnctic variation lor rcsistancc to Fusarium
car rot in ncld maizc (King and Scott, !98!, Clcmcnts ct al., 2004) but havc rcvcalcd no cvidcncc ol
complctc rcsistancc. A high gcnctic corrclation bctwccn car rot and lumonisin in two maizc populations
(Robcrtson ct al., 200) suggcsts that sclcction against car rot should rcsult in rcduccd susccptibility
to lumonisin contamination. Fumonisin contcnt has a highcr hcritability, so dircct sclcction against
lumonisin contcnt is prcdictcd to bc thcorctically morc cmcicnt than indircct sclcction against car rot
lor rcducing susccptibility to lumonisin contamination. Howcvcr, lumonisin assays rcquirc much morc
timc and moncy than car rot mcasurcmcnts. Tcrclorc, sclcction against car rot is hypothcsizcd to bc
morc practically cmcicnt at idcntilying lincs with rcduccd susccptibility to lumonisin.
A spccinc objcctivc ol this projcct is (!) to backcross gcncs conlcrring rcsistancc to Fusarium car rot
and lumonisin contamination lrom thc agronomically poor inbrcd linc G440 to thc susccptiblc clitc
linc FR!064, and (2) to tcst thc ccctivcncss ol sclcction against car rot at rcducing susccptibility to
lumonisin contamination in that population.
Quantitative Expression Analysis of Adversity Resistance Genes in Corn
Germplasm with Resistance to Preharvest Afatoxin Contamination
M. Luo
!,2
, . avis
1
, V. Xu
4
, . Lcc
2
, and 8.Z. Guo
!
!
2
University of Georgia,
Department of Crop and Soil Sciences, Tifton, GA;
1
University of Missouri-Columbia, Division of Plant
Sciences, Columbia, MO;
4
Texas A&M University, Lubbock, TX
Aatoxin contamination ol corn in thc ncld is known to bc inucnccd by numcrous lactors. rought
strcss is conducivc to Aspergillus favus inlcction and aatoxin accumulation. rought tolcrant gcrmplasm
could rcducc prcharvcst aatoxin contamination. Tc goals ol this projcct arc to undcrstand thc changcs
ol gcnc cxprcssion in rcsponsc to drought strcss using maizc microarray and to idcntily thc biochcmical
pathways and important gcncs associatcd with rcsistancc to A. favus and drought tolcrancc. !n this
rcport, wc arc rcporting thc dcvclopmcnt ol a sct ol gcnc/probcs in asscssmcnt ol maizc gcrmplasm with
drought tolcrancc and A. favus rcsistancc. !n our 2004 maizc microarray study, wc lound thc quantitativc
dicrcncc in gcnc cxprcssion undcr drought strcss, and thc rcsistancc by induction using 8TH was not
signincantly improvcd. 8ascd on thc gcnc cxprcssion analysis and rcportcd data, wc sclcctcd !!9 gcncs,
including two rclcrcncc gcncs, with advcrsity rcsistancc to tcst gcnc dicrcntial cxprcssion in six maizc
lincs, A618, 871, L!0!6, L964, M!7 and Tcx6, using rcaltimc RTPCR. Tc drought strcss was
applicd at 2AP (day altcr pollination) lor strcsscd plots. Corn cars at 1 AP wcrc harvcstcd and
only kcrncls wcrc uscd lor gcnc cxprcssion analysis in rcsponsc to drought strcss using thc dcsigncd
primcrs. Vc arc intcrcstcd in thc gcncs rclatcd with advcrsity rcsistancc, particularly drought tolcrancc
and lungal rcsistancc. Microarray is a powcrlul tool to sclcct important gcncs in rcsponsc to abiotic
and biotic strcsscs. For thc last two ycars, wc havc bccn using maizc microarray in scarching lor gcncs/
pathways associatcd with thcsc two traits and wc lound that thc dicrcntial cxprcssions ol thc majority
gcncs arc quantitativc. Tc rcaltimc RTPCR data ol thc sclcctcd gcncs indicatc that thc rcpcatability
ol this mcthod is high. Tc gcncs rclatcd with signal pathways had high C(T) cyclcs, and 86 gcncs
had dctcctablc and rcpcatablc changcs in rcsponsc to drought strcss or among thc sclcctcd maizc lincs.
A618, TX6 and L964 had morc uprcgulatcd gcncs in comparison with 871. !l 871, L!0!6 and
M!7 wcrc uscd as rclcrcncc lincs, rcspcctivcly, thcrc wcrc !0 crosstalking positivc gcncs which can
bc sclcctcd lrom A618, TX6 and L964. Tcsc gcncs arc rclatcd with drought rcsponsc and discasc
rcsistancc.
Peanut PR Protein, -1,3-glucanase, Induction by Aspergillus favus and
Copurifcation with a Conglutin-like Protein
X. Liang
!
, 8.Z. Guo
2
, and C.C. Holbrook
1
!
Guangdong Academy of Agricultural Sciences, Guangzhou, China;
2
Management Research Unit, Tifton, GA;
1
USDA-ARS, Crop Genetics and Breeding Unit, Tifton, GA
Aatoxin contamination ol pcanut has bccn idcntincd as thc most important hcalth problcm lacing thc
pcanut industry. !nlcction ol pcanut (Arachis hypogaea L.) sccds by Aspergillus favus and A. parasiticus
is a scrious problcm that can rcsult in aatoxin contamination in thc sccds. 8rccding rcsistant cultivars
would bc an ccctivc approach to rcducc aatoxin accumulation. Tc objcctivc ol this study was to
invcstigatc thc cxprcssion ol pathogcncsisrclatcd (PR) protcin !,1glucanasc and thc isolorm
pattcrns in pcanut sccds inoculatcd with A. favus. Pcanut gcnotypcs, GTC9 and GTC20 (both
rcsistant to Aspergillus favus inlcction), and Gcorgia Grccn and A!00 (both susccptiblc to A. favus
inlcction), wcrc uscd in this study. Tc activitics ol !,1glucanasc wcrc similar in thc uninlcctcd sccds
ol all gcnotypcs, but incrcascd signincantly in thc rcsistant gcnotypcs altcr inoculation in comparison
with thc susccptiblc gcnotypcs. An ingcl (nativc PAG) cnzymatic activity assay ol !,1glucanasc
rcvcalcd that thcrc wcrc morc protcin bands corrcsponding to !,1glucanasc isolorms in thc inlcctcd
sccds ol rcsistant gcnotypcs than in thc inlcctcd sccds ol susccptiblc gcnotypcs. 8oth acidic and basic
!,1glucanasc isolorms wcrc dctcctcd in thc !F gcl. Tin laycr chromatography (TLC) analysis ol
thc hydrolytic products lrom thc rcaction mixturcs ol thc substratc with thc total protcin cxtract or
individual band ol nativc PAG rcvcalcd thc prcscncc ol cnzymatic hydrolytic oligomcr products. Tc
individual bands corrcsponding to thc bands ol !,1glucanasc isolorms Glu ! wcrc scparatcd on
thc SSPAG rcsulting in two bands, !0ka and !1ka, rcspcctivcly. Tc scqucncc ol thc !1ka
major protcin band showcd a high dcgrcc ol homology to conglutin, a storagc protcin in pcanut sccds.
Conglutin is rcportcd as a pcanut allcrgcn, Ara h, and has trypsin inhibitor lunction. ur data providc
thc nrst cvidcnccs lor pcanut having !,1glucanasc activitics and thc association with thc rcsistancc to
A. favus colonization in pcanut sccds.
Corn Husk Characteristics Potentially Associated with Resistance to Afatoxin
Contamination of Grain: A Preliminary Study
M.J. Clcmcnts and V.P. Villiams
USDA-ARS, Corn Host Plant Resistance Research Unit, Mississippi State, MS
Fccding damagc lrom scvcral inscct pcsts on corn cars contributcs to conditions that lavor scvcrc
Aspcrgillus and Fusarium car rots and scvcrc mycotoxin accumulation in grain. Husk charactcristics that
scrvc as barricrs to inscct movcmcnt oltcn arc ncgativcly associatcd with inscct lccding damagc to cars,
and thcrclorc arc thought to scrvc as mcchanisms ol rcsistancc to discasc dcvclopmcnt and mycotoxin
accumulation in grain. Typically, husk tightncss has bccn quantincd subjcctivcly cithcr shortly altcr
silking or altcr plants havc dricd down and arc rcady lor harvcst. Rationalc lor timing ol husk tightncss
cvaluations is not availablc in litcraturc. ur objcctivc is to idcntily a plant growth stagc or scasonal
pcriod at which dicrcntiation ol husk tightncss among various corn gcnotypcs is maximizcd. Vc
cxamincd lour mcthods ol cvaluating husk tightncss ovcr six sampling pcriods in rcplicatcd trials at
Mississippi Statc Univcrsity in 200. Forcc rcquircd to rcmovc husk lcavcs lrom thc car was mcasurcd
mcchanically (gaugcd pull) and subjcctivcly (subjcctivc pull). Forcc rcquircd push a 1mm dia. stccl rod
longitudinally along thc car bctwccn husk lcavcs and kcrncls was mcasurcd mcchanically lrom thc tip to
thc shank cnd ol thc car (adhcsion lrom thc tip) and lrom thc shank to thc tip cnd ol thc car (adhcsion
lrom thc shank). Grcatcst dicrcntiation ol husk tightncss among gcnotypcs was obscrvcd bctwccn 28
to 1 days post midsilk lor thc lour mcthods cvaluatcd. Gaugcd pull at 28 days post midsilk providcd
grcatcst rangc ol data, and good rcsolution to dicrcntiatc thc lour gcnotypcs tcstcd, howcvcr, all
lour mcthods warrant lurthcr cxamination as potcntial mcthods ol quantilying husk tightncss. Tis
study will bc rcpcatcd in 2006 along with, potcntially, an cvaluation ol husk tightncss among F2:1
lamilics associatcd with rcsistancc or susccptibility to aatoxin contamination in grain in anothcr study.
!nlormation on timing ol husk tightncss cvaluations will optimizc studics aimcd at dicrcntiating
gcnotypcs with tight or loosc husks and thc idcntincation ol quantitativc trait loci associatcd with husk
tightncss.
Chalcone Synthase, a Gene that Infuences Both Drought Response and
Afatoxin Accumulation in Maize
M. Gcrau, . 8ush, . avis, C. Morriss, and G. avis
Division of Plant Sciences, University of Missouri-Columbia, Columbia, MO
rought strcss incrcascs aatoxin contamination in maizc. !dcntincation ol thc gcncs that mcdiatc this
ccct will lacilitatc dcvclopmcnt ol stablc, low aatoxin accumulation maizc lincs. Roots arc thc nrst
organ to scnsc soil drying. !dcntincation ol gcncs that arc involvcd in carly root rcsponsc to soil drying
is an important nrst stcp towards rcducing drought strcss and thc associatcd incrcascs in aatoxin
lcvcl. Towards this cnd, wc idcntincd thirtyscvcn root architccturc QTL in thc maizc !ntcrmatcd
871 Mo!7 mapping population. 8oth wcllwatcrcd and watcrstrcsscd root architccturc was studicd.
Scvcral ol thc QTL corrcspond to gcncs lor abscisic acid (A8A) biosynthcsis. A8A has long bccn
linkcd to drought rcsponsc. Abscisic acid lcvcls havc bccn implicatcd in mcdiating rcactivc oxygcn
spccics damagc. Prior QTL analysis ol aatoxin accumulation in maizc idcntincd a QTL that coincidcs
with thc chalconc synthasc gcnc in maizc. Chalconc synthasc (c) is a polykctidc sccondary mctabolitc
produccd by thc maizc plant which is thc ratc limiting stcp in anthocyanin pigmcnt production and
scrvcs as a branch point to scvcral othcr avonoid compounds. Subscqucntly, thc rolc ol this gcnc in
aatoxin was connrmcd in indcpcndcnt studics. Naringcnin, a product ol thc c gcnc has also bccn
shown to rcducc Aspergillus favus growth in vitro. Tc c gcnc has also bccn implicatcd in changcs in
root branching undcr watcr strcss by QTL and mutant analysis. Tis gcnc rcprcscnts a potcntial targct
lor rcducing drought strcss rclatcd incrcascs in aatoxin lcvcls and undcrstanding thc mcchanism by
which thcy occur. !n othcr plants, naringcnin has bccn shown to incrcasc branching by accting polar
auxin transport. Studics arc undcrway to dctcrminc whcthcr auxin is involvcd in mcdiating thc drought
associatcd incrcasc in aatoxin accumulation in maizc.
90
+8:n Axxu~i Avi~:oxix ii:ix~:iox Vovxsnov: Svssiox : 91
18
TH
SESSION 2: MICROBIAL ECOLOGY
Moderator: Phil Vakclyn, National Cotton Council
90
Efect of Fungal Competition on the Colonization of Wounded Peanut Seeds by
Aspergillus section Flavi from Natural Soil Populations
8.V. Horn
USDA-ARS, National Peanut Research Laboratory, Dawson, GA
Tc ccct ol lungal compctition on thc colonization ol woundcd pcanut sccds by Aspergillus scction Flavi
spccics in soil was cxamincd. \iablc pcanut sccds wcrc woundcd and inoculatcd with cultivatcd soils
dicring in composition and dcnsity ol Aspergillus spccics, thcn incubatcd lor !4 d at dicrcnt tcmpcraturcs
and sccd watcr activitics. Maximum pcrccntagcs ol sccd colonization by scction Flavi spccics occurrcd at
2217 C and a sccd watcr activity ol 0.920.96. Undcr thcsc conditions, compctitivc saprotrophic ability
ol scction Flavi was high and approximatcly 0 ol thc pcanut sccds with a propagulc ol A. favus or A.
parasiticus at thc wound sitc bccamc colonizcd. Vounds inoculatcd with soil wcrc initially colonizcd by
A. terreus (24 days), which was thcn quickly ovcrgrown by scction Flavi spccics and A. niger (> 4 days).
Furthcr succcssional changcs in thc pcanut mycobiota wcrc not obscrvcd cxccpt lor thc appcarancc ol
Eupenicillium ochrosalmoneum sporulating on thc hcads ol scction Flavi spccics. A signincant intcractivc
ccct (P < 0.000!) was obscrvcd bctwccn soil dcnsitics ol A. favus and soil dcnsitics ol othcr, potcntially
compcting spccics within scction Flavi (A. parasiticus, A. caelatus and A. tamarii). Colonization ol sccds
by A. favus dccrcascd as soil dcnsitics ol compcting scction Flavi incrcascd. Soil dcnsitics ol scction
Flavi spccics and A. niger showcd a similar intcractivc ccct (P < 0.000!). Tcrclorc, compctition
among Aspcrgilli is rcsponsiblc lor supprcssing sccd colonization by individual scction Flavi spccics.
thcr spccics in thc gcncra Aspergillus, Penicillium and Fusarium wcrc capablc ol invading pcanut sccds
primarily whcn soils containcd low dcnsitics ol scction Flavi spccics (< 0 CFU/g) or whcn combinations
ol tcmpcraturc and sccd watcr activity wcrc suboptimal lor scction Flavi.
92 +8:n Axxu~i Avi~:oxix ii:ix~:iox Vovxsnov: Svssiox :
Transfer of Afatoxin Biocontrol Technology: Results of First Commercial Use
in Peanuts
Joc V. orncr
USDA-ARS, National Peanut Research Laboratory, Dawson, GA
A mcthod lor biological control ol aatoxin contamination ol pcanuts has bccn dcvclopcd through
scvcral ycars ol rcscarch. 8iological control is achicvcd by introducing a dominant population ol a
nontoxigcnic strain ol Aspergillus favus into thc soil ol thc growing pcanut crop, and thc applicd strain
compctitivcly cxcludcs toxigcnic strains in thc colonization ol pcanuts during pcriods ol latcscason
drought. A signincant accomplishmcnt in thc dcvclopmcnt ol this tcchnology was thc dcvclopmcnt ol
a uniquc lormulation tcchniquc in which conidia ol thc nontoxigcnic strain arc coatcd onto thc surlacc
ol hullcd barlcy, which scrvcs as a carricr lor dclivcry ol thc lungus to thc ncld and also as a substratc lor
lurthcr prolilcration ol thc lungus altcr application. ARS patcnts lor this tcchnology wcrc liccnscd lor
commcrcialization in 2002, and thc biocontrol product, aaguard, rcccivcd PA scction 1 rcgistration
as a biopcsticidc in 2004. Tis madc possiblc thc nrst commcrcial usc ol thc product on approximatcly
000 acrcs ol pcanuts during crop ycar 2004 in southcastcrn Alabama and southwcstcrn Gcorgia. To
dctcrminc thc cmcacy ol aaguard in largcscalc usc, soil samplcs lrom rcprcscntativc trcatcd and
untrcatcd nclds wcrc dilution platcd to dctcrminc A. favus populations and toxigcnicity. !n addition,
larmcrs stock pcanut samplcs wcrc collcctcd at buying points in cach arca ol usc and analyzcd lor
aatoxin. Finally, trcatcd and untrcatcd pcanuts that had bccn storcd scparatcly lor scvcral months wcrc
analyzcd altcr shclling to dctcrminc aatoxin in shcllcd lots prior to salc.
Application ol aaguard changcd thc composition ol A. favus soil populations lrom 7!.!
toxigcnic strains in untrcatcd nclds to only 4.0 in trcatcd soils. Analyscs ol larmcrs stock pcanuts
bcing dclivcrcd at scvcn dicrcnt locations showcd a consistcnt rcduction in aatoxin contamination in
pcanuts lrom nclds trcatcd with aaguard. vcr all locations, aatoxin avcragcd 78.9 ppb in untrcatcd
pcanuts comparcd with !!.7 ppb in trcatcd pcanuts, an 8.2 rcduction. Pcanuts lrom trcatcd and
untrcatcd nclds wcrc storcd togcthcr in scparatc warchousc bins at two dicrcnt locations. Aatoxin
analyscs at thc Unadilla, GA location showcd a mcan aatoxin conccntration in all shcllcd cdiblc lots
lrom untrcatcd nclds ol 16.2 ppb comparcd with a mcan ol 0.9 ppb in lots lrom trcatcd nclds. At thc
awson, GA storagc location, aatoxin mcans lor shcllcd lots wcrc 7.2 and 2.2 ppb lor untrcatcd and
trcatcd pcanuts, rcspcctivcly. For shcllcd lots to bc sold to a manulacturcr, an omcial aatoxin analysis
ol thc lot must show thc lot to contain ! ppb ol aatoxin. !l thc lot contains > ! ppb, costly rcmilling
and blanching must bc carricd out to try to rcducc thc lcvcl to ! ppb or lcss. !n analyscs ol shcllcd
untrcatcd lots lrom thc Unadilla warchousc, 48.4 ol thosc lots tcstcd at > ! ppb comparcd with
no such lots lrom thc trcatcd pcanuts. At thc awson location, !.8 ol shcllcd lots lrom untrcatcd
nclds containcd > ! ppb comparcd with no lots ol trcatcd pcanuts. Tis translatcs to a rcduction in nct
shcllcd stock valuc lor untrcatcd pcanuts ol !1.0 and 4.1 lor Unadilla and awson, rcspcctivcly. Using
thc uropcan Union tolcrancc ol 4 ppb lor total aatoxins, thosc rcductions in valuc lor untrcatcd
pcanuts wcrc !9.! and !!.6, rcspcctivcly. Convcrting thc valuc changcs to a larmcrs stock ton basis, thc
dicrcnccs in nct larmcrs stock valuc lor thc pcanuts storcd in Unadilla wcrc 8!. and 872.78 pcr ton,
rcspcctivcly, lor thc ! and 4 ppb tolcranccs. quivalcnt dicrcnccs lor thc awson pcanuts wcrc 8!!.11
and 819.!8, rcspcctivcly. Tcsc data illustratc thc cconomic bcncnts that can bc gaincd lrom usc ol thc
biocontrol agcnt to control aatoxin in pcanuts.
Atoxigenic Strain Technology for Afatoxin Control in Cotton
Larry Antilla
!
and Pctcr J. Cotty
2
!
Arizona Cotton Research & Protection Council Phoenix, AZ;
2
Agricultural Research Service, USDA,
Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ
Atoxigcnic strains ol Aspergillus favus havc bccn invcstigatcd as biological control agcnts lor thc
mitigation ol aatoxin contamination ol a varicty ol commcrcial crops sincc thc latc !980s. nc such
strain, Aspergillus favus AF16, occurs naturally in Arizona soils and has rcpcatcdly bccn dcmonstratcd
to havc thc ability to compctitivcly cxcludc aatoxin producing lungi and thcrcby rcducc aatoxin
contamination ol cottonsccd. Tc Arizona Cotton Rcscarch & Protcction Council and thc Agricultural
Rcscarch Scrvicc cstablishcd a partncrship to dcvclop commcrcial scalc AF16 production and cvaluatc
commcrcial scalc mcthodologics lor utilizing atoxigcnic strains in thc latc !990s. Tc goals ol this
uniquc coopcrativc vcnturc wcrc to: a) dcvclop arcawidc managcmcnt stratcgics lor thc Arizona cotton
industry, b) obtain PA rcgistration ol AF16, thcrcby lacilitatc its maximum utilization on cotton, c)
systcmatically cvaluatc cccts ol variability in growcr utilization ol AF16 trcatmcnts on cmcacy in
aatoxin managcmcnt, and d) dcvclop standard opcrating proccdurcs and manulacturing protocols
which would allow othcr intcrcstcd growcr groups or coopcrativcs to rcadily acccss this public scctor
tcchnology. Ficld cvaluation studics wcrc thc subjcct ol incrcascd attcntion during thc 2004 trcatmcnt
scason. 8ascd on obscrvations lrom 2001, it appcarcd that light soils with rcduccd moisturc carrying
capacity had a tcndcncy to support poor growth ol AF16 and thcrcby poor cmcacy il applications wcrc
madc prior to adcquatc canopy closurc and hcncc rcduccd humidity lcvcls at thc soil surlacc. Studics
suggcstcd that dircct cxposurc ol inoculatcd whcat sccd to sunlight has a dclctcrious ccct on lungal
survival. At thc vcry lcast, sccds cxposcd to dircct sunlight wcrc in a moisturc dcprivcd cnvironmcnt and
thcrclorc lcss capablc ol sporulation. !n an cort to corrcct this situation in 2004, a tcst was arrangcd
with a coopcrating gin to maximizc conditions lor AF16 survival. Tc tcst involvcd thrcc participating
growcrs with acrcagc totaling 648. A scasonal cmploycc was hircd to coordinatc all trcatmcnts to cxact
program spccincations. All nclds wcrc trackcd to 80 canopy closurc or abovc. !rrigation schcdulcs
wcrc carclully rccordcd so that AF16 could thcn bc applicd by tractor using Gandy boxcs and drop
tubcs 24 hours or lcss prior to irrigation onsct. Post trcatmcnt cvaluations indicatcd that ncar !00
ol thc applicd AF16 product (colonizcd whcat sccd) sporulatcd. At harvcst, lourtccn nclds lrom thc
block wcrc tcstcd lor AF16 and thc highly toxigcnic S strain. AF16 displaccmcnt lcvcls on thc crop
rangcd lrom 7!!00 with an avcragc ol 82.4 lor all nclds. Corrcsponding S strain lcvcls rangcd
lrom 0 to 7. with an ovcrall avcragc ol 0.6. Tis rcprcscntcd a signincant changc in thc lungal
community ratio lor thc principal larmcr in thc tcst arca whcrc prctrcatmcnt background soil analyscs
on nvc nclds avcragcd 46 S strain and only onc pcrccnt AF16. Tc dramatic cccts associatcd with
incrcascd growcr attcntion to dctail during trcatmcnts with atoxigcnic strain AF16 cstablishcd thc
nccd lor a rctrospcctivc analysis ol commcrcial applicator practiccs in rclationship to thc highcst and
lowcst lcvcls ol displaccmcnt ol aatoxin produccrs by AF16 lollowing trcatmcnt to crops. To this cnd, a
standard qucstionnairc was dcvclopcd to charactcrizc and quantily produccr practiccs and obscrvations
associatcd with AF16 applications. Tc inlormation rcqucstcd lrom growcrs rclatcd to application,
irrigation, pcsticidc usc, crop charactcristics, and wcathcr and pcst conditions (bird, rodcnt and inscct).
!n addition, a soil typc databasc was initiatcd to lurthcr clucidatc thc rclationship bctwccn product
pcrlormancc and thc broad rangc ol cxtrinsic lactors inucncing such. Arcawidc program statistics
lor Arizona in 2004 arc as lollows: a total ol 21,419 acrcs wcrc trcatcd rcprcscnting twclvc scparatc
gcographical arcas in thc Statc. Tcn gins participatcd and a total ol 8! scparatc commcrcial nclds wcrc
trcatcd. A total ol 77 soil samplcs wcrc collcctcd and analyzcd with avcragc prctrcatmcnt AF16 and S
strain lcvcls ol 27 and 26, rcspcctivcly. A total ol !2 post application crop samplcs takcn at harvcst
2004 avcragcd 6 AF16 and 1 S strain. Arcas that cxpcricncc particularly largc bcncnts lrom AF16
continued next page
Managing Afatoxins in Cotton-Corn Rotations
Pctcr J. Cotty
USDA-ARS, Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ
applications continuc to bc thosc that trcat rclativcly largc acrcagc in thc samc gcographical arcas ovcr
multiplc ycars. Two such ginning communitics in Yuma County havc, through 2004, complctcd two or
morc ycars ol coordinatcd program activitics. Rcsults ol trcating morc than 7,900 acrcs combincd havc
produccd an avcragc AF16 lcvcl on thc crop ol 8!. with a corrcsponding S strain avcragc ol 4..
Tc ability ol AF16 to carry ovcr lrom onc crop ycar to thc ncxt in situations whcrc trcatmcnts arc not
applicd annually has bccn routincly dcmonstratcd in numcrous program scttings. Tc most striking
cxamplc ol this phcnomcnon was documcntcd in thc Mohavc \allcy in northwcstcrn Arizona in 2004.
AF16 trcatmcnts ol 910 and 2,700 acrcs in 2002 and 2001 rcspcctivcly produccd avcragc displaccmcnt
lcvcls ol 8! AF16 in thc soil and 8 on thc crop. No trcatmcnts wcrc madc in thc Mohavc \allcy
during 2004. cspitc thc lact that no Mohavc trcatmcnts wcrc madc in 2004, AF16 still composcd 82
ol thc A. favus rcsidcnt in Mohavc \allcy soils prcviously trcatcd. Modincd lungal community structurc
was maintaincd throughout thc 2004 crop scason and 68 ol thc A. favus on thc 2004 Mohavc \allcy
cottonsccd was AF16 altcr ginning. Futurc corts will locus on continucd charactcrization ol optimum
trcatmcnt dclivcry systcms and on improvcd manulacturing and lormulations.
Antilla & Cotty, continued
Afatoxin Control in Pistachios: Biocontrol Using Atoxigenic Strains
Mark ostcr
!
, Tcmis Michailidcs
!
, Pctcr Cotty
2
, avc Morgan
!
, Lorcnc 8occklcr
!
, an Fclts
!
,
and Hcraclio Rcycs
!
!
University of California/Kearney Agricultural Center, Parlier, CA;
2
Southern Regional Research Center,
ARS/USDA, New Orleans, LA
For thc past scvcral ycars, wc havc invcstigatcd thc usc ol atoxigcnic strains (strains not ablc to producc
aatoxins) ol Aspergillus favus as biocontrol agcnts to rcducc aatoxin contamination ol pistachios in
Calilornia. Tis approach has bccn vcry succcsslul in commcrcial cotton nclds in Arizona whcrc thc
atoxigcnic strain AF16 has substantially rcduccd aatoxin contamination ol cottonsccd. !n 200! and
2002, thc thrcc promising atoxigcnic strains A64, A8!, and AF16 (this strain, thc samc as uscd in
Arizona cotton nclds, was only applicd in 2002) wcrc applicd in a oodirrigatcd rcscarch pistachio
orchard. !n carly summcr lor both ycars, whcat sccds inlcctcd with thcsc strains wcrc applicd to thc
orchard oor at thc ratc cquivalcnt to !0 lbs/acrc. No atoxigcnic strains havc bccn applicd in this
orchard sincc 2002. !n ordcr to dctcrminc thc survival and sprcad ol thc atoxigcnic strains, soil samplcs
wcrc collcctcd on 10 August, 2004. Tc dcnsity ol A. favus/A. parasiticus in thc soil did not signincantly
dicr among trcatmcnts. Most ol thc A. favus isolatcs lrom thc soil in thc trcatcd arcas bclongcd to
thc atoxigcnic strain applicd thcrc (80.1 to 96.7 ol thc isolatcs, dcpcnding on strain) cvcn though thc
atoxigcnic strains had not bccn applicd sincc 2002, dcmonstrating that thc atoxigcnic strains pcrsist
wcll in pistachio orchard soil. Tc applicd strains wcrc dctcctcd in thc untrcatcd arcas at low lcvcls (2.4
to 4.9 ol thc isolatcs, dcpcnding on strain), suggcsting only slight movcmcnt ol thc applicd atoxigcnic
strains to untrcatcd arcas. !n 200 additional soil samplcs wcrc collcctcd on 29 August and arc currcntly
bcing cvaluatcd.
Starting in 2001, thc atoxigcnic strain AF16 was applicd in a dicrcnt rcscarch pistachio orchard
that was irrigatcd by microsprinklcrs. !n 2004 whcat sccds inlcctcd with AF16 wcrc applicd altcr
collccting soil samplcs on 6 July. n !2 Scptcmbcr, samplcs ol lcavcs, carly split nuts, and soil wcrc
collcctcd. n thc lollowing day, thc nuts wcrc harvcstcd, and samplcs collcctcd lrom thc harvcstcd
nuts. !n latc summcr thc dcnsity ol A. favus/A. parasiticus in soil was not signincantly dicrcnt bctwccn
trcatcd arcas and untrcatcd arcas. Tc incidcncc ol AF16 among A. favus isolatcs incrcascd lrom bclorc
applying thc whcat to latc summcr in thc trcatcd arcas (lrom 0.6 to 2.8 and lrom 42.4 to 70.!
lor arcas trcatcd lor onc ycar and two ycars, rcspcctivcly) but not in thc untrcatcd arcas (6.! and !.!).
Tc incidcncc ol AF16 on lcavcs did not signincantly dicr bctwccn arcas trcatcd with AF16 (!9.!
and 27.2) and untrcatcd arcas (!7.). No kcrncl dccay by A. favus was lound in ovcr 600 carly split
nuts, suggcsting that applying AF16 docs not signincantly incrcasc dccay ol thc nuts. !n addition, thc
trcatmcnts did not dicr signincantly in thc dcnsity ol A. favus/A. parasiticus on thc surlacc ol thc hulls
ol lrcshly harvcstcd nuts. !n 200 whcat inlcctcd with AF16 was applicd on 29 Junc altcr collccting soil
samplcs. Lcal, nut, and additional soil samplcs wcrc collcctcd in latc summcr ol 200 and arc currcntly
bcing cvaluatcd.
Tc incidcncc ol atoxigcnic strains among A favus isolatcs occurring naturally in commcrcial
pistachio orchards in Calilornia was dctcrmincd. All thrcc atoxigcnic strains, AF16 (.1), A64 (!.7),
and A8! (!.0) wcrc dctcctcd among 794 isolatcs ol A. favus lrom commcrcial pistachio orchards. A
ncw study was initiatcd that will dctcrminc thc natural incidcncc ol ! additional atoxigcnic A favus
strains in commcrcial pistachio orchards. Prcliminary rcsults show that thcsc othcr atoxigcnic strains
occur at vcry low lcvcls in commcrcial pistachio orchards.
Afatoxin Control in Figs: Biocontrol and New Resistant Cultivars
Mark ostcr
!
, Tcmis Michailidcs
!
, Pctcr Cotty
2
, Louisc Fcrguson
!
, Jamcs oylc
!
, avid
Morgan
!
, Lorcnc 8occklcr
!
, an Fclts
!
, and Hcraclio Rcycs
!
!
University of California, Davis/Kearney Agricultural Center, Parlier, CA;
2
Southern Regional Research
Center, ARS/USDA, New Orleans, LA
For scvcral ycars, wc havc invcstigatcd thc usc ol atoxigcnic strains (strains not ablc to producc aatoxins)
ol Aspergillus favus as biocontrol agcnts to rcducc aatoxin contamination ol ngs in Calilornia.
Tis approach has bccn vcry succcsslul in commcrcial cotton nclds in Arizona whcrc thc atoxigcnic
strain AF16 has substantially rcduccd aatoxin contamination ol cottonsccd. !n 2004 wc applicd thc
atoxigcnic strain AF16 lor a sccond ycar in a dripirrigatcd Calimyrna ng orchard. n 6 July, whcat
sccds inlcctcd with AF16 wcrc applicd at thc ratc ol 4!.2 g whcat/trcc (cquivalcnt to !0 lbs/acrc). n 26
August, wc collcctcd noncaprincd ngs lrom thc orchard oor. As in 2001, thc applicd atoxigcnic strain
AF16 was dctcctcd colonizing and sporulating on thc noncaprincd ngs. n ! Scptcmbcr, lcal, lruit,
and additional soil samplcs wcrc collcctcd. Tc soil had a highcr dcnsity (!07.1 clu/g soil) ol A. favus/A.
parasiticus in thc arcas undcr thc drip lincs whcrc inlcctcd whcat had bccn placcd than in thc middlcs
(!!.0 clu/g) or undcr thc drip lincs in thc untrcatcd arcas (0.1 clu/g). !n latc summcr, all ol thc A. favus
isolatcs obtaincd lrom thc soil undcr thc drip lincs in thc arcas trcatcd in 2001 and 2004 bclongcd to
thc strain AF16 comparcd to only 47.1 ol thc isolatcs lrom thc untrcatcd arcas. Furthcrmorc, thc
incidcncc ol AF16 in thc middlcs was 92.1, suggcsting that thcrc was movcmcnt ol AF16 lrom thc
applicd arcas undcr thc drip lincs to thc middlcs. Tc dcnsity ol A. favus/A. parasiticus and thc incidcncc
ol AF16 on lcavcs did not dicr signincantly bctwccn trcatmcnts. No dccay by A. favus was lound in
900 dricd ngs that wcrc cxamincd, suggcsting that applying AF16 docs not signincantly incrcasc dccay
ol thc ngs. ur rcsults suggcst that thc usc ol AF16 in ng orchards should rcsult in thc atoxigcnic strain
bccoming thc dominant A. favus strain whcrc applicd without signincantly incrcasing ng dccay.
!n 200 wc did not apply any atoxigcnic strains in this orchard. Howcvcr, wc did collcct samplcs in
ordcr to dctcrminc thc survival and sprcad ol thc prcviously applicd atoxigcnic strains. n 26 August,
wc collcctcd noncaprincd ngs lrom thc orchard oor but obscrvcd no colonics ol A. favus on thcm. n
7 Scptcmbcr, lcal, ng, and soil samplcs wcrc collcctcd and arc currcntly bcing cvaluatcd.
Tc incidcncc ol atoxigcnic strains among A. favus isolatcs occurring naturally in commcrcial
ng orchards in Calilornia was dctcrmincd. A total ol 122 isolatcs ol A. favus lrom commcrcial ng
orchards wcrc cvaluatcd, and all thrcc atoxigcnic strains AF16 (7.8), A64 (0.6), and A8! (0.6)
wcrc dctcctcd. !n addition, a ncw study was initiatcd that will dctcrminc thc natural occurrcncc ol !
additional atoxigcnic A. favus strains in commcrcial ng orchards.
Ncw ng sclcctions havc bccn dcvclopcd by a Univcrsity ol Calilornia brccding program that produccd
sclcctions having Calimyrna anccstry but with ngs that had smallcr cycs (thc opcning to thc intcrior
ol thc ng) than Calimyrna ngs and did not nccd to bc pollinatcd by thc ng wasp. Tc most promising
sclcction, prcviously namcd 618V but now givcn thc namc Sicrra, has bccn rclcascd to commcrcial
ng growcrs. !n 2004 Sicrra ngs had substantially smallcr cyc diamctcr ol dricd lruit (!. mm) than thc
commcrcial cultivars Adams (2.1 mm) and Calimyrna (2.2 mm) but thc samc as Conadria (!. mm).
Furthcrmorc, thc incidcncc ol dccay by Aspergillus scct. Flavi ol thc dricd ngs ol Sicrra was substantially
lowcr (0.0) than that ol Calimyrna (.7). Tc ngs ol thc ncw ng cultivar Sicrra havc consistcntly had
substantially lcss lungal dccay, including dccay by aatoxinproducing lungi, than Calimyrna ngs.
Identifcation of Bacterial Antagonists of Aspergillus favus from California
Almond Orchards
Jcrcy . Palumbo, Jamcs L. 8akcr and Norccn . Mahoncy
USDA-ARS,Western Regional Resarch Center, Albany, CA
8actcrial populations lrom two Calilornia almond orchards wcrc cvaluatcd lor thcir potcntial as
biological control agcnts against Aspergillus favus. 8actcria wcrc isolatcd lrom washcs ol almond
owcrs, immaturc lruits and maturc lruits by dircct plating. !solatcd strains wcrc scrccncd lor
antagonistic activitics against A. favus strain Papa827, a nor mutant strain that accumulatcs norsolorinic
acid undcr aatoxigcnic conditions. !nhibition ol growth and aatoxin production (visualizcd by thc
orangc pigmcntation ol norsolorinic acid) by bactcrial isolatcs was asscsscd using agar and liquid
bascd coculturc assays. !nitial scrccns idcntincd 118 isolatcs with antilungal phcnotypcs and wcrc
studicd lurthcr. l thcsc, !47 isolatcs inhibitcd growth ol A. favus, and 24 isolatcs inhibitcd aatoxin
production. Tcsc isolatcs wcrc lurthcr charactcrizcd by cxamining thcir production ol cxtraccllular
chitin and ycast ccll wallhydrolyzing cnzymc activitics. 8actcrial strains wcrc idcntincd by !6S rNA
scqucncc analysis and by nutritional analysis using thc 8iolog microbial idcntincation systcm. Scvcral
gcncra wcrc idcntincd, including Bacillus, Pseudomonas, Ralstonia, Burkholderia, and scvcral plant
associatcd cntcric and noncntcric bactcria. 8ccausc ol thcir rclativc lrcqucncy ol isolation, as wcll
as thcir antilungal activitics and rcsistancc to cnvironmcntal strcss, Bacillus isolatcs appcar to bc most
promising lor dcvclopmcnt lor biocontrol ol A. favus on almonds.
Biological Control of Aspergillus favus by a Saprophytic Yeast Strain in Tree-
Nut Orchards: Progress in 2005
Sui Shcng Hua
USDA-ARS, Western Regional Research Center, Albany, CA
Tc lungus, Aspergillus favus, produccs aatoxin which is thc most potcnt carcinogcn known. Tis
mycotoxin is vcry hazardous to thc hcalth ol both human and animal. National cconomic losscs
arc in thc billions ol dollars pcr ycar duc to aatoxin contamination ol agricultural commoditics
including pistachio. Rcsistant pistachio hybrids arc not availablc. Tus biological control is a viablc and
cnvironmcntallricndly approach to managc prcharvcst aatoxin contamination.
Tc ycast, Pichia anomala strain VRL076 inhibitcd aatoxin production ol A. favus by 99 in a
bioassay protocol (Hua ct al., Appl nviron Microbiol !999, 6: 27182740). Tis particular ycast was
dcmonstratcd to rcducc sporc production ol both toxigcnic and aatoxigcnic isolatcs ol A. favus in
pistachio owcrs and nutlruits as wcll as in almond and pistachio lcavcs in thc lab cxpcrimcnts (Hua,
Acta Hort 2002, 9!: 2710, Hua, !8C 8ullctin 2004, 27: 29!294).
Tc saprophytic lungus, A. favus, inlccts plants through wounds. Aatoxin contamination is wcll
documcntcd to bc associatcd with wounding in corn, pcanuts, cotton and trccnuts bclorc harvcst. Two
cxpcrimcnts wcrc conductcd in a commcrcial orchard in thc summcr ol 200 in collaboration with
.. Parntt and 8rcnt Holtz, Univcrsity ol Calilornia avis. Nutlruits ol pistachio wcrc individually
woundcd with a dissccting nccdlc. Four trcatmcnts wcrc applicd. 8ranchcs ol nut clustcrs wcrc spraycd
with watcr, spraycd with an aqucous suspcnsion ol ycasts at !0 cclls/ml, spraycd with an aqucous
suspcnsion ol ycasts at !0 cclls/ml and two hours latcr spraycd with sporc suspcnsion ol A. favus
at ! !0` cclls/ml, or spraycd with a sporc suspcnsion ol A. favus at ! !0` cclls/ml. Four trccs wcrc
randomly sclcctcd lor cach trcatmcnt. Nutlruits wcrc harvcstcd 1 wccks altcr spraying. Tc data
show that P. anomala VRL076 rcduccd thc lrcqucncy ol A. favus colonization by 4 to !0 timcs and
dccrcascd thc total propagulcs ol A. favus by 80 to 99 in comparison to nutlruits not spraycd with
thc ycast.
Vc dcmonstratcd that P. anomala could grow at low watcr activity (a
w
). PG (polycthylcnc glycol)
8000 was uscd to adjust mcdium a
w
to 0.96, which mimickcd a watcr strcss condition ol .62 MPa.
Tc ycast cclls lormcd a nlm and inhibitcd thc growth ol A. favus inoculatcd to thc mcdium. Aatoxin
contaminations ol corn, pcanut, cotton and trcc nuts arc known to bc associatcd with watcr strcss. Tis
trait makcs thc spccics vcry suitablc as a biological control agcnt against A. favus undcr watcr strcss
conditions.
Scvcntccn mcdia ol dicrcnt composition wcrc cvaluatcd lor thc production ol P. anomala to gcncratc
high numbcrs ol ycast cclls. Tc bioassay protocol was uscd to asscss cach mcdium lor thc production ol
ycast cclls compctcnt in inhibiting thc growth and aatoxin production ol A. favus. Two mcdia showcd
good yicld ol ycast cclls and supportcd thc production ol compctcnt P. anomala lor biological control
ol A. favus.
Cultural Conditions Promoting Chitinase Production in Gliocladium
catenulatum
avid F. Kcndra
!
, Michacl J. Muhitch
2
, Ambcr Andcrson
!
and Ccsaria . McAlpin
!
!
Mycotoxin Research Unit, USDA, ARS, National Center for Agricultural Utilization Research, Peoria, IL;
2
Rochester College, Rochester Hills, MI
Gliocladium catenulatum is a known mycoparasitc ol scvcral lungal gcncra including Aspergillus and
Fusarium. Tc usc ol mycoparasitcs to control lungal plant pathogcns cithcr as dircct biocontrol agcnts
or as novcl sourccs ol antilungal compounds is incrcasing in importancc to scrvc as cnvironmcntal
altcrnativcs to chcmical control. Tc antagonistic activity ol biocontrol agcnts is attributablc to onc
or morc complcx mcchanisms including thc production ol antibiotic mctabolitcs, compctition lor
nutricnts, induction ol systcmic acquircd rcsistancc, incrcascd nutricnt availability to thc host plant
and production ol ccll wall hydrolyzing cnzymcs, including chitinasc and !,1glucanasc. Gliocladium
catenulatum is known to producc scvcral sccondary mctabolitcs with antimicrobial activity, including
vcrticillin and glisoprcnin howcvcr thcrc is no rcport ol chitinolytic activity. !n this papcr wc rcport thc
cultural conditions rcquircd to producc chitinasc in minimal mcdia supplcmcntcd with chitin. ptimal
production ol cxtraccllular chitinasc was obscrvcd in a liquid mcdium prcviously dcvclopcd lor culturing
Fusarium chlamydosporum whcn culturcd at 2 C lor 2! days and a slightly acidic pH. Chitinasc activity
was rcprcsscd il thc colloidal chitin mcdium was amcndcd with sucrosc, glucosc, ccllulosc or starch
and climinatcd by xylan or lactosc. Tc nitrogcn sourcc signincantly inucnccd chitinasc activity with
KN supporting thc bcst production.
PANEL DISCUSSION: Microbial Ecology
Panel Chair: 8rucc Horn
Panel Members: L. Antilla, P.J. Cotty, J.V. orncr, M. ostcr, S.S. Hua, .F. Kcndra,
J.. Palumbo
Jcrcy Palumbo was askcd to addrcss a potcntial problcm with biological control in almond orchards,
namcly, that biocontrol bactcria and aatoxinproducing lungi havc dicrcnt tcmpcraturc and watcr
activity rcquircmcnts. Hc rcplicd that most ol thc work thus lar has bccn pcrlormcd in thc laboratory
and that luturc ncld trials should indicatc how dicrcnt cnvironmcntal conditions acct thc ability ol
bactcria to inhibit aatoxin production.
Larry Antilla was askcd how carly application ol nonaatoxigcnic A. favus can bc ccctivc il thc
cotton canopy is not lully dcvclopcd. Hc rcplicd that application is carly in thc Yuma rcgion ol Arizona
bccausc canopy closurc is a month ahcad ol cottongrowing rcgions lurthcr to thc wcst. A lair amount
ol canopy and associatcd shading arc rcquircd lor maximum sporulation ol thc biocontrol lungus. Pctcr
Cotty addcd that at 6 rclativc humidity during thc pcriod ol application (middlc ol Junc to thc middlc
ol July), !820 moisturc in thc whcat grain containing thc biocontrol lungus is dimcult to attain. Soil
typc is also a lactor: cotton canopy may bc morc important in sandy soils than in clay soils, which arc
bcttcr at rctaining moisturc. Furthcrmorc, nativc toxigcnic populations ol A. favus arc incrcasing in soil
during canopy lormation and il thc biocontrol application is dclaycd too long, ccctivc displaccmcnt
ol toxigcnic strains may not bc possiblc. onald Vicklow mcntioncd that A. favus sclcrotia applicd
to Gcorgia crop nclds gcrminatcd undcr thc crop canopy but not in thc cxposcd rcgions bctwccn thc
rows.
Pctcr Cotty was askcd to dcscribc thc latc ol highly contaminatcd cottonsccd altcr arrival at thc oil
mill. Hc statcd that whcn thc sccds arc crushcd, aatoxins arc rctaincd in thc mcal and arc not prcscnt
in thc oil. Howcvcr, a prontablc oil mill must markct thc highvaluc mcal, which is rich in protcin. Mills
oltcn scgrcgatc sccds bclorc crushing to kccp aatoxin conccntrations in mcal bclow 20 ppb, sccd lots
that cxcccd this limit arc markctcd in Mcxico. Tc hulls ol cottonsccd typically do not contain aatoxins
but during proccssing, thcy may bccomc contaminatcd with aatoxins lrom dust and sccd lragmcnts.
Joc orncr was askcd il thcrc was any inccntivc lor growcrs to usc biocontrol in pcanuts. Hc rcplicd that
thcrc is littlc inccntivc lor thc growcrs undcr thc currcnt pcanut program bccausc larmcrs stock pcanuts
arc cxamincd lor visiblc A. favus but arc not analyzcd lor aatoxins. vcn il aatoxins arc prcscnt, thc
growcr is not pcnalizcd bccausc pcanuts without visiblc A. favus (scg !) havc a guarantccd govcrnmcnt
loan pricc rcgardlcss ol aatoxin conccntration. Scg ! pcanuts arc latcr analyzcd lor aatoxins altcr
thcy arc shcllcd and sizcd in commcrcial shclling plants. Tcrclorc, most ol thc cconomic burdcn lor
aatoxins is bornc by thc shclling companics. Tcrc is somc cvidcncc that aaguard
supprcsscs othcr
pcanut lungal discascs such as whitc mold (Sclerotium rolfsii). An incrcasc in pcanut yicld rcsulting lrom
rcduccd discasc incidcncc may providc inccntivc lor pcanut growcrs to utilizc biocontrol.
avid Kcndra was askcd about thc chitosan uscd in his cxamination ol chitinasc production by
Gliocladium catenulatum. Hc rcplicd that chitosan is dcacctylatcd chitin and that thc amount ol
acctylation on thc backbonc ol thc molcculc, an important lcaturc in thc activity ol chitinasc, can bc
rcgulatcd. Chitosan can bc maintaincd at a high molccular wcight with varying dcgrccs ol acctylation.
Crab shcll chitin, in contrast, contains impuritics and rcquircs considcrablc clcan up.
Tc pancl in gcncral was askcd whcthcr biocontrol lungi inducc rcsistancc in crop plants. Sylvia Hua
answcrcd that thc induction ol rcsistancc gcnc products altcr spraying corn silks with ycast might bc
dctcctcd through analysis ol mcsscngcr RNA and protcin products. 8rucc Horn addcd that bccausc
crops arc invadcd by A. favus at low watcr activitics, phytoalcxins which rcquirc high watcr activitics
would not bc an important lorm ol rcsistancc. Joc orncr also mcntioncd that inlcction lcvcls rcmain
thc samc in untrcatcd and trcatcd crops and, thcrclorc, an incrcasc in host rcsponsc would not bc
cxpcctcd.
Mark ostcr was askcd whcthcr cyclopiazonic acid (CPA) has cvcr bccn dctcctcd in pistachio nuts.
Hc rcplicd that CPA has not bccn dctcctcd but that thc nuts havc only rcccntly bccn cxamincd lor
this mycotoxin. 8ccausc CPAproducing lungi, such as Aspergillus tamarii, occur in pistachio nuts, low
lcvcls might bc cxpcctcd. !n a lollowup qucstion conccrning thc corrclation bctwccn high aatoxin
contamination and lowyicld ycars lor pistachio nuts, ostcr statcd that naval orangcworm damagc is
morc cxtcnsivc during lowyicld ycars, which may account lor incrcascd aatoxin contamination.
Pctcr Cotty was askcd whcthcr populations ol A. favus S strain would rcbound il applications ol
nonaatoxigcnic A. favus wcrc discontinucd. Hc rcplicd that nclds rcquirc continuous trcatmcnt.
Stablc biocontrol populations cannot bc maintaincd bccausc ol thc largc inux ol toxigcnic strains
lrom surrounding nclds. Sincc thc S strain oltcn compriscs 70 80 ol A. favus populations in soils,
this particular morphotypc is wcll adaptcd to its cnvironmcnt. Anothcr qucstion poscd lor Cotty
conccrncd thc rcasons why sorghum docs not bccomc highly contaminatcd with aatoxins. Sorghum
has a complicatcd mycobiota and thc high incidcncc ol cmatiaccous lungi may compctitivcly inhibit
invasion by aatoxinproducing lungi. !n arcas ol Alrica whcrc both sorghum and corn arc plantcd, corn
bccomcs highly contaminatcd with aatoxins whcrcas sorghum contains low lcvcls ol thc mycotoxin.
For thc nnal topic ol discussion by thc pancl, Joc orncr was askcd about thc proccdurcs lor sampling
pcanuts lor aatoxins. !n unshcllcd larmcrs stock pcanuts, a 2kg samplc is rcmovcd and cxamincd
only lor visiblc A. avus. Tcsc samplcs show cxtrcmc variability in aatoxin lcvcls whcn tcstcd lor
cxpcrimcntal purposcs. Altcr shclling, pcanuts arc analyzcd lor aatoxins by rcmoving thrcc 48pound
samplcs lrom cach 22ton lot. !n thc nrst samplc, pcanuts that tcst lor aatoxins at 8 ppb and > 4
ppb arc acccptcd and rcjcctcd, rcspcctivcly, without lurthcr tcsting. !l thc aatoxin conccntration lalls
within this rangc, thc sccond samplc is analyzcd. !l thc mcan ol nrst and sccond samplcs is !2 ppb,
thc pcanut lot is acccptcd, il thc mcan is > 21 ppb, thc lot is rcjcctcd. Finally, il thc mcan aatoxin lcvcl
lalls within this rangc, thc third samplc is analyzcd and thc mcan ol all thrcc samplcs must bc ! ppb
lor acccptancc. A lollowup qucstion conccrncd thc latc ol pcanut lots that havc bccn rcjcctcd bccausc
ol aatoxin contcnt. Tc shcllcr may havc thc sccds blanchcd and rccolor sortcd. Rcmoval ol thc skins
during blanching cxposcs discoloration in pcanuts and as a conscqucncc, color sorting bccomcs morc
ccctivc. !l thc blanchcd pcanuts still lail thc aatoxin analysis, thc sccds arc uscd lor oil.
Infuences of Crops and Geographic Features on Communities of Afatoxin-
producing Fungi
Ramon Jaimc
!
and Pctcr J. Cotty
2
!
Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ;
2
USDA-ARS, Division
of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ
Aatoxins arc produccd by ascxual lungi bclonging to Aspergillus scction Flavi. Soils in arcas whcrc
contamination is common contain divcrsc communitics ol aatoxin producing lungi. Communitics ol
scction Flavi dicr by rcgion in both spccics composition and aatoxin producing potcntial. A. favus can
bc dividcd into two morphotypcs (thc S and L strains) bascd on morphological, gcnctic, and physiologic
critcria. Tc strain S produccs numcrous small sclcrotia (avcragc diamctcr < 400 m) and high lcvcls ol
aatoxins, whilc thc strain L produccs lcwcr, largcr sclcrotia (avcragc diamctcr > 400 m) and variablc
lcvcls ol aatoxin, ranging lrom nonc to high lcvcls. Tc strain S ol A. favus has bccn rcportcd as a
natural soil inhabitant in scvcral arcas worldwidc including Southcast Asia, South Amcrica and North
Amcrica. Sincc cottonsccd is a prclcrrcd lccd lor dairy cows and aatoxin 8 in lccd is translcrrcd to thc
milk in thc slightly modincd lorm aatoxin M, dairics typically pay a prcmium lor clcan cottonsccd.
Tus, in arcas whcrc aatoxin contamination is common, aatoxin contcnt is thc most important lactor
dctcrmining sccd valuc. 8oth A. favus community structurc and aatoxin contamination prcscnt spatial
variation. Howcvcr, rclationships ol contamination to lungal community structurc in soils havc not
bccn dcscribcd. Tc objcctivcs ol thc currcnt study wcrc to: !) spatially analyzc A. favus communitics in
soils ol South Tcxas, 2) cvaluatc inucnccs ol crop rotation on community structurc ol A. favus, and 1)
dctcrminc rclationships ol A. favus community structurc to soil tcxturc.
Tc structurc ol A. favus communitics rcsiding in soils ol South Tcxas was dctcrmincd by analyzing
126 soil samplcs lrom !2 nclds locatcd lrom thc Rio Grandc \allcy in thc south to Fort 8cnd county
in thc north in thc springs ol 200! to 2001. Tc prcvious scason crop was idcntincd lor most samplcd
nclds. Soil samplcs wcrc ovcn dricd at 48 C lor 48 hours bclorc proccssing. A. favus was isolatcd lrom
soil by dilution plating onto a modincd Rosc 8cngal agar. Aspergillus scction Flavi colonics wcrc sub
culturcd on /2 agar ( \8 juicc and 2 agar) lor to 7 days at 1! C and assigncd cithcr to thc A. favus
S or L strains, A. tamarii or A. parasiticus on thc basis ol colony charactcristics and isolatc morphology.
Colony lorming units (CFU/g), Pcrccnt ol thc S strain (Pcrccnt S), and pcrccntagcs ol clay, silt and
sand wcrc analyzcd by Analysis ol \ariancc and \ariancc Componcnts Analysis using Gcncral Lincar
Modcls, Pcarsons Corrclation Analysis, and gcostatistics.
Aspergillus favus communitics in soils ol South Tcxas dicr signincantly among rcgions in both
community dcnsity (CFU/g) and S strain (Pcrccnt S) incidcncc. Quantitics ol A. favus wcrc grcatcr in
arcas ol thc Uppcr Coast than in arcas ol thc Costal 8cnd. n thc othcr hand, avcragc S strain incidcncc
was lowcr in arcas ol thc Rio Grandc \allcy than in arcas ol cithcr thc Coastal 8cnd or Uppcr Coast.
Small gcographic scalcs (within and among nclds) cxplaincd most ol thc variancc lor CFU/g (91.9)
and 68 ol thc variancc lor Pcrccnt S, whilc largc gcographic scalcs (among rcgions) only inucnccd
Pcrccnt S cxplaining 24 ol thc variancc. Most ol thc variation (7.9 to 90.7) lor thc soil tcxturc
variablcs clay, silt and sand occurs among nclds and among arcas. Ficlds whcrc thc prcvious crop was
corn had highcr CFU/g (!,48) comparcd to cithcr cotton (66) or sorghum (!7). n thc othcr hand,
nclds prcviously croppcd to cotton had highcr Pcrccnt S (28.6) than thosc to corn (!7.0). Ficlds
prcviously croppcd to sorghum wcrc intcrmcdiatc bctwccn thosc croppcd to cotton and corn. Pcarsons
corrclation analyscs showcd signincant positivc corrclations bctwccn pcrccnt ol clay and both Pcrccnt
S and CFU/g, and signincant ncgativc corrclation bctwccn pcrccnt ol sand and both Pcrccnt S and
CFU/g. Tc rcsults ol this study dcmonstratc that both crop rotation and soil tcxturc inucncc both
population dcnsity ol A. favus and incidcncc ol thc S strain.
Afatoxin Contamination of Maize in Africa
Claudia Probst
!
, Hcnry Njapau
2
, and Pctcr J. Cotty
!,1
!
Division of Plant Pathology and Microbiology, e University of Arizona, Tucson, AZ;
2
Center for Food
Safety and Applied Nutrition, Food and Drug Administration, College Park, MD;
1
USDA-ARS, e
University of Arizona, Tucson, AZ
Aatoxins arc carcinogcnic and tcratogcnic mctabolitcs produccd by scvcral Aspergillus spccics,
including Aspergillus favus and Aspergillus parasiticus, during inlcction ol a varicty ol crops cithcr prior
to or altcr harvcst. Aatoxinproducing lungi vary widcly in many charactcristics and both ability to
inlcct and dccay crops and aatoxinproducing capacity dicr among aatoxinproducing lungi. Tus,
thc potcntial ol thcsc lungi to contaminatc crops with aatoxin also varics. ctcrmining thc most
important causcs ol a contamination cvcnt rcquircs considcring both thc aatoxinproducing potcntial
ol thc lungi prcscnt and thc lrcqucncics with which thcy occur in thc contaminatcd crop. Maizc (Zea
mays) is highly susccptiblc to inlcction by aatoxinproducing lungi. Such inlcctions can lcad to scvcrc
contamination with aatoxin, rcsulting in diminishcd crop valucs and incrcascd hcalth risks lor animals
and humans. Ncgativc hcalth conscqucnccs lor humans causcd by ingcstion ol aatoxin contaminatcd
loods includc impaircd growth, canccr and dcath. Many countrics havc sct maximum allowablc lcvcls
lor aatoxins in ordcr to limit hcalth risk. Howcvcr, thcsc standards havc littlc inucncc on ingcstion ol
aatoxins by most poor, smallscalc larmcrs in Alrica.
uring January to Junc 2004, 1!7 cascs ol acutc aatoxicosis in astcrn and Ccntral provinccs ol
Kcnya wcrc idcntincd, with a casclacility ratc ol 19. Tc cpidcmic was causcd by ingcstion ol maizc
with aatoxin conccntrations up to 4,400 ppb. Although aatoxins havc bccn associatcd with lcthal
lood poisoning in Kcnya thrcc timcs sincc !98!, thc lungi contaminating thc maizc with aatoxins havc
not bccn charactcrizcd. Vc analyzcd !01 maizc samplcs collcctcd during thc 2004 outbrcak lrom thc
most acctcd districts in ordcr to idcntily thc most important aatoxinproducing lungi. A total ol
!,221 Aspergillus Scction Flavi isolatcs wcrc rccovcrcd lrom thc maizc and charactcrizcd. vcr 97 ol
thc Scction Flavi isolatcs wcrc A. favus and thc rcmindcr (2.1) wcrc A. parasiticus. No othcr aatoxin
producing lungi wcrc dctcctcd. A. favus can bc dclincatcd into two morphotypcs, thc S and L strains,
which dicr in lruiting habit and aatoxinproducing ability. Tc majority (71) ol thc A. favus isolatcs
bclongcd to thc S strain, which was not prcviously known in Alrica. S strain isolatcs produccd much
grcatcr quantitics ol aatoxins than thc L strain isolatcs (66 g aatoxin 8/g mycclium, n-!!7, vcrsus
40 g aatoxin 8/g mycclium, n-10). l thc lungi cxamincd, only A. parasiticus produccd G aatoxins.
8oth thc S and L strain A. favus produccd only 8 aatoxins. !ncidcncc ol thc S strain incrcascd with
avcragc aatoxin contcnt lrom 69 in samplcs with <20 ppb total aatoxins to 94 in samplcs with
> !,000 ppb total aatoxin. !ndccd, thc S strain occurrcd in thc poisonous maizc at highcr proportions
than prcviously obscrvcd on any crop lrom any location, worldwidc. Tc distinct ccology ol thc S strain
should bc takcn into account during dcvclopmcnt ol mcthods to prcvcnt luturc aatoxicoscs in Kcnya.
Tis causal agcnt should bc thc targct ol longtcrm prcvcntativc mcasurcs.
Infuences of Herbicides on Release of Atoxigenic Strains
Nicholas P. Garbcr
!
and Pctcr J. Cotty
2
!
Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ;
2
USDA-ARS, Division
of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ
Screening of Atoxigenic Aspergillus favus Isolates for Ability to Inhibit Afatoxin
B Production by Toxigenic Aspergillus favus
A. Jha, R. Swcany and K.. amann
Dept. of Plant Pathology & Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA
8iological control ol aatoxigcnic Aspergillus favus using atoxigcnic isolatcs ol thc samc lungus has
bccn dcmonstratcd in cotton and pcanuts. !n ordcr to sclcct potcntial biocontrol isolatcs lor corn, a
collcction ol 4! atoxigcnic isolatcs ol A. favus wcrc individually cvaluatcd lor ability to inhibit aatoxin
8 production by a singlc toxigcnic isolatc in a suspcndcd disc assay. ight isolatcs complctcly inhibitcd
aatoxin production whcrcas 4 othcrs wcrc highly inhibitory. Tcsc sclcctcd isolatcs will bc applicd to
corn in a ncld tcst to dctcrminc thcir ability to prcvcnt aatoxin contamination.
106
+8:n Axxu~i Avi~:oxix ii:ix~:iox Vovxsnov: Svssiox 107
18
TH
SESSION 3: CROP RESISTANCE GENETIC ENGINEERING
Moderator: Kccrti Rathorc, Tcxas A&M Univcrsity
106
Gene-based Antifungal Strategies in Peanut
Yc ( Julict) Chu
!
, Paola Faustinclli
!
, Laura Ramos
!
, Kanniah Rajasckaran
2
, Jc Cary
2
, Corlcy
Holbrook
1
, Pcggy ziasAkins
!

!
Department of Horticulture, University of Georgia Tifton Campus, Tifton, GA;
2
USDA-ARS, SRRC, New
Orleans, LA;
1
USDA-ARS, Coastal Plain Experiment Station, Tifton, GA
Gcnctic mcchanisms lcading to antilungal stratcgics lor aatoxin rcduction in pcanut arc bcing invcstigatcd.
Tcsc includc gcnctic cnginccring as wcll as mutation brccding approachcs. Pcanut gcnctic cnginccring is
rclativcly incmcicnt comparcd with many othcr crops, tcsting lor aatoxin rcduction in thc ncld takcs
scvcral ycars, and acccptancc ol transgcnic crops is tcnuous in somc parts ol thc world. Tcrclorc, in addition
to antilungal stratcgics bascd on gcnctic cnginccring, wc arc placing incrcasing cmphasis on spccinc gcnc
mutations that could acct rcsistancc traits. Trcc objcctivcs havc bccn thc locus ol our rcscarch ovcr thc
past ycar. Tc nrst has bccn to charactcrizc transgcnic pcanut containing an antiapoptotic gcnc lrom
human, 8clxl. Tc sccond has bccn to cxplorc thc usc ol a mutant population gcncratcd lor discovcry ol
gcnc lunction lor aatoxin rcduction goals. Tc third has bccn to tcst allcrgcn gcnc promotcr ability to drivc
cxprcssion ol rcportcr gcncs.
Tc putativc antilungal gcnc 8clxl cncodcs lor an antiapoptotic protcin lrom humans. 8clxl has bccn
translormcd into tobacco and was shown to conlcr broadspcctrum lungal pathogcn rcsistancc as wcll
as rcsistancc to tomato spottcd wilt virus (ickman ct al., PNAS 200!, 98: 697). Altcr microprojcctilc
bombardmcnt ol cmbryogcnic culturcs, wc rccovcrcd 42 indcpcndcnt hygromycinrcsistant lincs ol pcanut
lrom which 109 plants wcrc rcgcncratcd. Almost 90 (269) wcrc PCR positivc lor 8clxl. Scvcral lincs havc
bccn tcstcd by wcstcrn blotting lor protcin cxprcssion and by RTPCR lor RNA cxprcssion. Plants that
arc PCR positivc lor prcscncc ol thc gcnc also havc bccn RTPCR positivc indicating that cxprcssion at
thc RNA lcvcl was occurring. Fcwcr lincs havc shown dctcctablc protcin cxprcssion cithcr as a rcsult ol low
cxprcssion lcvcls or low scnsitivity ol thc wcstcrn. nc linc that produccd progcny and was scgrcgating lor
thc transgcnc was cxamincd in morc dctail. !t has bccn spcculatcd that 8clxl can lunction as a strcssrclatcd
protcin, thcrclorc, wc havc tcstcd thc tolcrancc ol translormcd plants to paraquat as a quick assay lor protcin
activity. nc progcny plant that consistcntly showcd cxprcssion ol 8clxl on wcstcrn blots also consistcntly
showcd a highcr lcvcl ol tolcrancc to paraquat than cithcr thc background gcnotypc, Gcorgia Grccn, or
thc progcny that no longcr containcd thc transgcnc. thcr plants that showcd cxprcssion according to
RTPCR rcsults, but inconsistcnt wcstcrn blot rcsults, wcrc cithcr intcrmcdiatc in tolcrancc to paraquat
or similar in rcsponsc to thc nontransgcnic control. !t is possiblc that a thrcshold lcvcl ol cxprcssion is
rcquircd lor cmcacious allcviation ol strcss duc to hcrbicidc trcatmcnt.
For thc sccond objcctivc ol mutation brccding, thc goal is to producc a T!LL!NG population that can bc
scrccncd lor lipoxygcnasc mutants or any othcr gcnc suspcctcd to cnhancc aatoxin production. T!LL!NG
stands lor Targcting !nduccd Local Lcsions !N Gcnomcs (McCallum ct al., Plant Physiol 2000, !21:
419), is a mutation stratcgy, and was nrst tcstcd in Arabidopsis. T!LL!NG can idcntily mutants bascd on
scrccning with gcnc scqucncc rathcr than lor phcnotypc. Lcal samplcs havc bccn collcctcd lrom ~!00 M2
plants mutagcnizcd with cthylmcthanc sullonatc and NA has bccn cxtractcd lrom 184 ol thcsc to usc
lor T!LL!NG. Tc T!LL!NG tcchniquc is bcing tcstcd with an allcrgcn gcnc, ara h , lor which wc havc
gcncratcd sumcicnt gcnomic scqucncc lor this purposc. Two copics ol thc ara h gcnc arc prcscnt in pcanut,
onc lrom thc A gcnomc and onc lrom thc 8 gcnomc. Gcncspccinc primcr scts havc bccn dcsigncd lor
T!LL!NG so that mutations in cach copy ol ara h can bc scrccncd scparatcly.
Charactcrization ol allcrgcn gcnc scqucncc has allowcd thc isolation ol promotcrs that may bc usclul
lor antilungal gcnc cxprcssion, particularly whcn cxprcssion is to bc targctcd to thc dcvcloping sccd. Tc
allcrgcn promotcr scqucnccs havc bccn luscd with a rcportcr gcnc (glucuronidasc or GUS) and tcstcd lor
transicnt cxprcssion altcr bombardmcnt ol immaturc cotylcdons. GUS cxprcssion was obscrvcd with both
a ! kb and 2 kb upstrcam lragmcnt lrom ara h . Tc 2 kb lragmcnt contains a mcthyl jasmonatc rcsponsc
clcmcnt, thcrclorc, thc ccct ol this signaling molcculc on cxprcssion ol thc gcnc is bcing studicd.
108 +8:n Axxu~i Avi~:oxix ii:ix~:iox Vovxsnov: Svssiox
Transgenic Peanuts with Enhanced Resistance to Aspergillus favus
Arthur K. Vcissingcr
Department of Crop Science, North Carolina State University, Raleigh, NC
Abstract not availablc. Plcasc scc pagc !6 ol thc introduction lor a summary.
Identifcation, Characterization and Antifungal Activities of Silk Proteins in
Aspergillus favus Resistant and Susceptible Corn Inbreds
8cla Pccthambaran
!
, Gary L. Vindham
2
, Lcigh Hawkins
2
, Paul Villiams
2
and awn S.
Luthc
!
!
Biochemistry and Molecular Biology Department, Mississippi State University, MS;
2
USDA-ARS, Corn
Host Plant Resistance Unit, MS
Rcscarch in our laboratory is locuscd on climinating aatoxin contamination in maizc (Zea mays L.)
by incrcasing rcsistancc to Aspergillus favus inlcction during car dcvclopmcnt. 8ccausc it has bccn
postulatcd that thc lungus cntcrs thc car via thc silks, wc arc invcstigating thc protcomc ol silk protcins
in maizc inbrcds that arc rcsistant or susccptiblc to aatoxin contamination and /or A. favus inlcction.
Vc hopc to idcntily protcins that dircctly contributc to thc rcsistancc phcnotypc or protcins/gcncs
that can bc uscd lor markcrassistcd sclcction in brccding programs. Control silks wcrc collcctcd lrom
Mp1!1, Mp420 (rcsistant), Tx60! (intcrmcdiatc rcsistancc) and Sc2!2M, Mp119 (susccptiblc) 2!
and 2 days altcr silk cmcrgcncc (AS). !nlcstcd cars wcrc inoculatcd with A. favus at ! (AS) and
wcrc collcctcd 2! AS and 2 AS. Silk protcins wcrc cxtractcd and analyzcd by 2dimcnsional gcl
clcctrophorcsis (2). Gcl imagcs wcrc analyzcd by P Qucst soltwarc (8ioRad) and comparison
wcrc madc among inbrcd and bctwccn inoculatcd and uninoculatcd samplcs. MAL!TF mass
spcctroscopy and LC/MS/MS wcrc uscd to idcntily common silk protcins and thosc that consistcntly
dicrcd among rcsistant and susccptiblc lincs, or inoculatcd and uninoculatcd cars. Sclcctcd candidatc
gcncs scqucnccs wcrc invcstigatcd lor polymorphism and its RNA cxprcssion was also studicd. Agar
platc assays using GFPtaggcd A. favus wcrc uscd to study thc rcsistancc potcntial ol protcins cxtractcd
lrom thc rcsistant and susccptiblc gcnotypcs. Tc growth ol A. favus in thc prcscncc ol thc silk protcin
cxtracts was dctcrmincd by mcasuring GFP uorcsccncc and crgostcrol contcnt.
Silencing the Expression of RAP Genes in Maize and the Efect on Host
Resistance against Aspergillus favus Infection and Afatoxin Production
ZhiYuan Chcn
!
, Robcrt L. 8rown
2
, Tomas . Clcvcland
2
, and Kcnncth amann
!
!
Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton
Rouge, LA;
2
Southern Regional Research Center, USDA-ARS, New Orleans, LA
Aatoxins arc carcinogcns produccd mainly by Aspergillus favus during inlcction ol susccptiblc crops
such as maizc (Zea mays L.). Although rcsistant maizc gcnotypcs havc bccn idcntincd, thc incorporation
ol rcsistancc into commcrcial lincs has bccn slow duc to thc lack ol sclcctablc markcrs. Rcccntly,
rcsistanccassociatcd protcins (RAPs) havc bccn idcntincd by comparing constitutivc protcin pronlcs
bctwccn rcsistant and susccptiblc maizc gcnotypcs using protcomics. Prcliminary charactcrization ol
somc ol thcsc RAPs suggcst that thcy play a dircct rolc in host rcsistancc, such as pathogcncsisrclatcd
protcin !0 (PR!0), or an indircct rolc, such as glyoxalasc !, through cnhancing thc host strcss tolcrancc.
Howcvcr, dircct cvidcncc lor thcir involvcmcnt in kcrncl rcsistancc wcrc lacking.
!n thc prcscnt study, an RNA intcrlcrcncc (RNAi) gcnc silcncing tcchniquc was uscd to silcncc thc
cxprcssion ol thcsc gcncs. RNAi silcncing is a posttranscriptional, scqucnccspccinc RNA dcgradation
proccss. !t is triggcrcd by a doublc strandcd (ds) RNA, lcading to thc dcgradation ol homologous RNA
cncodcd by cndogcnous gcncs, and transgcncs. A binary vcctor containing all thc kcy clcmcnts nccdcd
to gcncratc a dsRNA structurc was constructcd using Gatcway tcchnology. Two invcrtcd rcpcats ol
parts ol thc coding rcgion ol glx-I and pr- wcrc intcgratcd into thc vcctor through sitcspccinc
rccombination. Tc rcsulting constructs (GLX! RNAi vcctor and PR!0 RNAi vcctor) wcrc thcn
translormcd into immaturc maizc cmbryos using both bombardmcnt and Agrobacterium inlcction.
Tirty two out ol 18 and !! ol ! callus cloncs ol, glx-I and pr-, rcspcctivcly, rcprcscnting indcpcndcnt
translormation cvcnts wcrc connrmcd to bc positivc lor translormation through PCR. Tc cxtcnt ol
gcnc silcncing in transgcnic callus tissucs varics lrom onc to anothcr ranging lrom 20 to ovcr 99, and
dcpcnds on thc RNAi constructs bascd on rcaltimc PCR. !t appcars that callus cloncs gcncratcd lrom
PR!0 RNAi vcctor had morc dramatic intcrlcrcncc in thc cxprcssion (with an avcragc ol ovcr 90
silcncing) than that obscrvcd in callus cloncs rcgcncratcd lrom GLX! RNAi vcctor (with an avcragc
ol 0 silcncing). Tc RNAi silcnccd transgcnic maizc sccds havc also bccn obtaincd lrom plants
rcgcncratcd lrom Agrobacterium translormcd callus cloncs. Tc numbcr ol kcrncls pcr car also varics
signincantly, lrom as lcw as 2 to as many as !96. For cach construct, kcrncls lrom 8 cars wcrc gcrminatcd
and gcnomic NA was isolatcd. PCR connrmation ol translormation using sccdling gcnomic NAs
lound that only two out ol 8 and onc out ol 8 wcrc ncgativc, lor GLX! and PR!0, rcspcctivcly. Kcrncl
scrccning assay ol thc transgcnic maizc kcrncls dcmonstratcd a signincant incrcasc in susccptibility to
A. favus colonization and aatoxin production in somc ol silcnccd transgcnic lincs comparcd with non
silcnccd control kcrncls, suggcsting thcir dircct involvcmcnt in aatoxin rcsistancc in maizc.
Genetic Engineering of Cotton for Resistance to Phytopathogens including
Aspergillus favus
Kanniah Rajasckaran
!
, Mauricio Ulloa
2
, 8ob Hutmachcr
1
, Jc Cary
!
, Jcssc M. Jayncs
4
and
Tomas Clcvcland
!
!
USDA, ARS, SRRC, New Orleans, LA;
2
USDA-ARS, Western Integrated Cropping Systems Research Unit,
Cotton Enhancement Program, Shafter, CA;
1
UC Davis, CA;
4
Tuskegee University, Tuskegee, AL
Fcrtilc, transgcnic cotton plants cxprcssing thc synthctic antimicrobial pcptidc, 4!, wcrc produccd
through Agrobacteriummcdiatcd translormation (Rajasckaran ct al. 200). PCR products and Southcrn
blots connrmcd intcgration ol thc 4! gcnc, whilc RTPCR ol cotton RNA connrmcd thc prcscncc ol
4! transcripts. In vitro assays with crudc lcal protcin cxtracts lrom T0 and T! plants connrmcd that
4! was cxprcsscd at sumcicnt lcvcls to inhibit thc growth ol Fusarium verticillioides and Verticillium
dahliae comparcd to cxtracts lrom ncgativc control plants translormcd with p8!d1SuidAnos
(CGUS). Although in vitro assays did not show control ol prcgcrminatcd sporcs ol Aspergillus favus,
bioassays with cotton sccds in situ or in planta, inoculatcd with a GFPcxprcssing A. favus, indicatcd that
thc transgcnic cotton sccds inhibitcd cxtcnsivc colonization and sprcad by thc lungus in cotylcdons and
sccd coats. In planta assays with thc lungal pathogcn, ielaviopsis basicola, which causcs black root rot
in cotton, showcd typical symptoms such as black discoloration and constriction on hypocotyls, rcduccd
branching ol roots in CGUS ncgativc control T! sccdlings, whilc transgcnic T! sccdlings showcd a
signincant rcduction in discasc symptoms and incrcascd sccdling lrcsh wcight, dcmonstrating tolcrancc
to thc lungal pathogcn. Ficld cvaluation ol T2 progcny lor Fusarium wilt (Fusarium oxysporum l.sp.
vasinfectum (F\) Atk. Sny & Hans) racc ! was carricd out in sandy soil that also cxhibitcd prcscncc ol
rootknot ncmatodcs (Meloidogyne incognita). R2 progcnics ol lour indcpcndcnt translormation cvcnts
cxprcssing thc antilungal pcptidc 4!, a transgcnic control cntry with thc GUS markcr gcnc and
thc original nontransgcnic varicty (Cokcr 1!2), along with commcrcial Acala (G. hirsutum) and Pima
(G. barbadense) cultivars wcrc includcd in thc ncld cvaluation. ntrics wcrc plantcd in a randomizcd
complctc block dcsign with lour rcplications on !0 lcct long plots. Plant survival ratc, loliagc damagc
symptoms, vascular root staining, prcscncc ol rootknot, and agronomic data havc bccn collcctcd lor
thcsc cntrics. Prcliminary obscrvations indicatcd that thc transgcnic cntrics showcd a hcalthy, highcr
gcrmination stand (up to 68) than thc controls (41). Vc hopc to complctc thc initial ncld cvaluation
this ycar (200) and thc promising lincs will bc rctcstcd lor pathogcn rcsistancc including prcharvcst
rcsistancc to Aspergillus favus.
Rajasckaran K, Cary JV, Jayncs JM, Clcvcland T. iscasc rcsistancc conlcrrcd by thc cxprcssion ol a gcnc cncoding a
synthctic pcptidc in transgcnic cotton (Gossypium hirsutum L.) plants. Plant 8iotcchnology Journal 200, 1: 44.
PANEL DISCUSSION: Crop Resistance Genetic Engineering
Panel Chair: Arthur Vcissingcr
Panel Members: ZY Chcn, P. ziasAkins, 8. Pccthambaran, K. Rajasckaran
Pancl summary not submittcd duc to illncss ol thc pancl chair. Plcasc scc pagc !6 ol thc introduction
lor an ovcrvicw ol thc scssions prcscntations.
113
18
TH
SESSION 4: CROP MANAGEMENT AND HANDLING, INSECT CONTROL AND
FUNGAL RELATIONSHIPS
Moderator: Pat Lcary, Cotton !ncorporatcd
Update on Validation and Distribution of a Computer Program for Predicting
Mycotoxins in Midwest Corn
Patrick F. owd
USDA, ARS, National Center for Agricultural Utilization Research, Crop Bioprotection Research Unit,
Peoria, IL
Sincc thc last rcport, thc prcdictivc computcr program as bccn lurthcr validatcd in 2001 and 2004.
A corrclation cocmcicnt ol 0.8, altcr onc outlicr was rcmovcd, was obtaincd lor commcrcial ncld
samplcs lrom 20002001 (which includcd a lcw dozcn dicrcnt nclds and hybrids). Samplcs wcrc
prcdictcd to havc low lcvcls ol lumonisin in 2004 (lcss than ! ppm) and this is what occurrcd in all nclds
samplcd. No aatoxin was prcdictcd to occur in cithcr 2001 or 2004, and nonc was lound. !n 200, thc
program prcdictcd a high probability that A. favus inoculum would bc prcscnt at silking, which was
communicatcd to larmcrs prcscnt at a ncld day in latc Junc. Continucd prcdictions indicatcd low lcvcls
(lrom 10 to lcss than 20 ppb), dcpcnding on wcathcr conditions at dicrcnt locations. !nscct lcvcls wcrc
vcry low throughout most ol thc scason. ars in dry arcas wcrc monitorcd by both PF and popcorn
company rcprcscntativcs, and also brought in to clcvators lor aatoxin dctcrminations by larmcrs.
Kcrncls with 8GYF, and in onc casc, visiblc A. favus colonization, wcrc cncountcrcd. A mccting with
clcvator opcrators indicatcd somc samplcs wcrc tcsting positivc lor aatoxin at 20 ppb lrom scvcral
dicrcnt arcas, and scattcrcd rcjcctcd loads occurrcd through latc Scptcmbcr. Samplcs wcrc takcn in
nclds undcr study, and rcsults ol analyscs arc pcnding. As indicatcd prcviously, coopcration bctwccn
USA and !llinois Ccntral Collcgc rcsultcd in a Vindows vcrsion with an addcd Hclp scction, and a
custom modulc that allows onc to customizc il outlicr hybrids arc cncountcrcd providcd prior ycars
data, including mycotoxin lcvcls, arc availablc. A bcta vcrsion has bccn dcmonstratcd via nctwork
conlcrcnccs to dicrcnt companics (contact Katc Hara, Tcchnology !nlormation mccr, to arrangc
a dcmonstration: katcCncaur.usda.gov). An additional cconomic dccisionmaking modulc is partly
writtcn. Additional plans arc to validatc thc program lor popcorn, which is widcly grown in Ccntral
!llinois (samplcs wcrc takcn in 200) and othcr loodgradc corn as inlormation bccomcs availablc.
A nnalizcd vcrsion ol thc initial program may bc put on a wcbsitc within thc ncxt ycar or two, but
commcrcial intcrcst may altcr this schcdulc. vcrall, hybrids and cultural managcmcnt prcscntly uscd
by larmcrs, couplcd with morc rcccntly idcntincd lactors, such as usc ol 8t vcrsions ol prclcrrcd hybrids,
carly planting to cscapc catcrpillar damagc in milk stagc, scouting lor damaging insccts including usc ol
lurcs and traps idcntincd in projcct rcscarch, lungal monitoring using lcal axil matcrial, and thc prcdictivc
computcr program indicating whcn mycotoxinproducing lungi occur, or spccinc mycotoxin lcvcls that
may occur without intcrvcntion by thc larmcr, could bc rationally combincd into a managcmcnt plan.
Mechanisms of Preharvest Afatoxin Contamination in Peanut Infected by
Root-Knot Nematodes
Patricia Timpcr
!,
Corlcy Holbrook
!
, and avc Vilson
2
!
USDA ARS, Tifton, GA;
2
Department of Plant Pathology, University of Georgia, Tifton GA
!nlcction ol pcanut by rootknot ncmatodcs (Meloidogyne arenaria) can lcad to an incrcasc in aatoxin
contamination ol kcrncls whcn thc plants arc subjcctcd to drought strcss during pod maturation. !t
is not clcar whcthcr thc incrcascd aatoxin contamination is primarily duc to grcatcr invasion ol thc
gallcd pods by toxigcnic Aspergillus spp. or whcthcr root galling is also involvcd. Ncmatodc damagc
to thc pods and/or roots may also dclay pod maturity. Small, immaturc pcanuts arc morc pronc to
aatoxin contamination than arc maturc, undamagcd pcanuts. ur objcctivcs wcrc: !) to dctcrminc
thc contribution ol root and pod galling causcd by rootknot ncmatodcs to thc incrcasc in aatoxin
contamination, and 2) whcthcr ncmatodc inlcction incrcascs thc pcrccntagc ol immaturc pcanuts.
A grccnhousc cxpcrimcnt was conductcd in which pods and roots wcrc physically scparatcd. Pod
sct was rcstrictcd to soilnllcd pans (4! cm dia. !0 cm dcpth), whilc thc roots grcw undcrncath
thc pan into a pot. Rootknot ncmatodcs (RKN) wcrc applicd to thc root zonc ol hall thc plants, thc
othcr plants did not rcccivc ncmatodcs in thc root zonc. Plants rcccivcd thrcc pod trcatmcnts altcr
pod sct: onc application, two applications, and no application ol RKN. Tc trcatmcnts wcrc arrangcd
in a complctcly randomizcd dcsign with !2 rcplicatcs/trcatmcnt. Conidia ol Aspergillus favus and A.
parasiticus wcrc addcd to cach pan whcn thc plants startcd to owcr. Plants wcrc subjcctcd to drought
strcss 40 days bclorc harvcst. Tc rcsults wcrc similar among thc two trials ol thc cxpcrimcnt so thc data
was combincd lor analysis. Adding ncmatodcs to thc pod zonc had no ccct on aatoxin conccntrations
in thc pcanut kcrncl. Howcvcr, thc lack ol an ccct may havc bccn to duc to thc low occurrcncc ol
galling on thc pcanut hulls. !n pots whcrc ncmatodcs wcrc addcd to thc root zonc, 0 to 80 ol thc root
systcm was gallcd. Adding ncmatodcs to thc root zonc incrcascd (P - 0.001) aatoxin conccntrations in
thc pcanut kcrncls lrom 17 ppb in thc control to 67 ppb.
A ncld microplot study was conductcd in 2001 and 2004 to dctcrminc whcthcr inlcction ol pcanut
by RKN incrcascs thc pcrccntagc ol immaturc kcrncls. Hall ol thc !2 plots wcrc inoculatcd with
ncmatodcs at two dicrcnt timcs (at plant and altcr pcgging) and thc othcr hall wcrc not inoculatcd
with ncmatodcs. All plots wcrc inoculatcd with A. favus/A. parasiticus. rought was induccd to 6
wccks bclorc digging. !n both 2001 and 2004, thc prcscncc ol ncmatodcs did not incrcasc aatoxin
conccntrations in thc pcanuts. !n 2001, plants inlcctcd with RKN produccd a grcatcr (P - 0.000!)
pcrccntagc ol immaturc kcrncls than uninlcctcd plants, howcvcr, in 2004 ncmatodcs had no ccct on
thc pcrccntagc ol immaturc kcrncls cvcn though root galling and yicld rcductions wcrc grcatcr in 2004
than in 2001.
!n summary, inlcction ol pcanut roots by thc pcanut rootknot ncmatodc incrcascs aatoxin
contamination ol thc kcrncls. Ncmatodc damagc to thc roots rcsults in grcatcr drought strcss which
may rcsult in grcatcr susccptibility to aatoxin contamination. Tc contribution ol pod galling and
immaturc kcrncls on thc incrcasc in aatoxin lcvcls in ncmatodcinlcctcd pcanut arc still unclcar.
Experimental Use of the Pear Ester Kairomone to Improve Codling Moth
Control in Walnuts
.M. Light, K.M. Rcynolds, P. 8ouyssounousc, and 8.C. Campbcll
USDA-ARS, Western Regional Research Center, Plant Mycotoxin Research Unit, Albany, CA
Aspergillus invasion ol trcc nuts is primarily through inscct damagc by moth larvac. ur goal is to
diminish inscctcauscd nut damagc through thc usc ol novcl, spccicsspccinc control systcms bascd on
hostplant kairomoncs. 8ccausc adult lcmalc moths lay cggs that hatch into damaging larvac, controlling
both lcmalc codling moth adults and thc hatchcd larvac would crcatc grcatcr control cmcacy. Vc havc
idcntincd a singlc compound isolatcd lrom pcars, cthyl (2E, 4Z)2,4dccadicnoatc, that is a powcrlul
kairomonc, attracting both malc and lcmalc codling moth (CM) adults and ncwlyhatchcd, nconatc
larvac. Trough a coopcrativc rcscarch and dcvclopmcnt agrccmcnt, and an approvcd patcnt and liccnsc,
bctwccn USA/ARS and Trc, !nc., a global rcscarch program has bccn undcrway lor nvc ycars to
dcmonstratc possiblc control uscs lor thc kairomonc compound. Trc, !nc. pctitioncd and attaincd
both xpcrimcntal Usc Pcrmits and Rcscarch Authorizations lrom thc PA and thc Calilornia cpt.
Food & Agriculturc lor thc cxpcrimcntal application ol thc kairomonc in walnut orchards.
\arious control tactics arc bcing invcstigatcd using thc pcarcstcr kairomonc to dircctly managc both
adult and larval CM in walnut orchards. Tc primary control tactics bcing rcscarchcd arc using thc
kairomonc to augmcnt mating disruption ol malc moths and attract and kill baitsprays targcting
nconatc larvac. uc to thc implcmcntation ol thc Food Quality Protcction Act ol !996 thc most ccctivc
and incxpcnsivc insccticidcs lor codling moth control, thc organophosphatc (P) insccticidcs, will in
thc vcry ncar luturc bc highly rcstrictcd or complctcly banncd lrom usc. Also, thc currcnt altcrnativc
control matcrials, both inscct growth rcgulating (!GR) and biologicalviral insccticidcs and phcromonc
mating disruptants, and thcir rcquircd application ratcs arc much highcr or prohibitivc in cost. Tus,
both insccticidal and phcromonc mating disruption altcrnativc stratcgics must bc madc morc ccctivc,
aordablc, and acccptablc lor control usc. ur goal and hypothcsis is that thc pcarcstcr kairomonc will
act to improvc thc control cmcacy and diminish thc amount ol insccticidc and phcromonc disruptant
rcquircd to control damaging populations ol CM in walnut orchards. Tis is bascd on our prior rcportcd
rcscarch that mating disruption bc morc ccctivc using thc combination ol phcromonc and kairomonc
ovcr thc currcnt phcromoncalonc tactic. Also, nconatc CM larvac arc highly attractcd to thc kairomonc,
thus baitsprays ol kairomonc + insccticidc might attract kill targct larvac morc ccctivcly.
Trc, !nc. has dcvclopcd a microcncapsulatcd (MC) sprayablc lormulation ol thc kairomonc. Tc
sprayablc MCkairomonc was tankmixcd as an adjuvant with rcduccd ratcs ol insccticidcs and applicd
as a lullcovcragc spray by handgunspraycrs. Four insccticidcs tcstcd wcrc: two Ps choropyrilos and
phosmct, an !GR mcthoxylcnozidc and a CM granulosis virus. 8aitspray trials wcrc conductcd in
a 20 acrc walnut orchard, using a complctcly randomizcd block dcsign ol cight singlctrcc rcplicatcs
pcr trcatmcnt. Trcatmcnts wcrc thc insccticidcs alonc comparcd with trcatmcnts ol insccticidc + MC
adjuvant. Six application sprays wcrc applicd ( Junc midScptcmbcr). Controls wcrc 20 random pickcd
unspraycd trccs. ccurrcncc and dcgrcc ol nut damagc was cvaluatcd by nut drop, prcharvcst canopy
inlcstation, and harvcst nut knockdown sampling. Rcsults wcrc vcry cncouraging, with thc MC
kairomonc adjuvant rcducing CM damagc by 81 and 90 lor thc P insccticidcs and 4 and 47
lor thc !GR and viral insccticidcs. Navcl orangcworm damagc was also signincantly rcduccd.
Tcsc studics show promisc that thc kairomonc can improvc insccticidc cmcacy and contributc to
ncw !PM tactics lor CM and NV and thc intcgratcd rcduction ol aatoxin incidcncc.
Liberty Link and Urea on Afatoxin and Fumonisin Levels in Corn
H. Arnold 8runs and H. K. Abbas
USDA-ARS-MSA, Crop Genetics and Production Research Unit, Stoneville, MS
An cxpcrimcnt was donc to asccrtain il Libcrty hcrbicidc [glulosinatcammonium |2amino4(hydro
xymcthylphosphinyl)ammonium salt|] or urca |C(NH)| would rcducc lungal growth ol Aspergillus
favus Link cx Frics and Fusarium verticillioides (Sacc.) Nircnbcrg (synonym - F. moniliforme J. Shcld.),
and thus thcir rcspcctivc mycotoxins, aatoxin and lumonisin, in prcharvcst corn (Zea mays L.). Four
corn hybrids, two gcnctically modincd to bc rcsistant to Libcrty hcrbicidc and two nongcnctically
modincd, wcrc plantcd at Stoncvillc, MS in 200!, 2002, 2001, and 2004 in a randomizcd complctc
block with a splitplot arrangcmcnt ol trcatmcnts rcplicatcd lour timcs. Tc cxpcrimcnt was lurrow
irrigatcd. !ndividual plots wcrc two rows 9 m long, spaccd !02 cm apart, and includcd onc ol thc
lollowing trcatmcnts: !) untrcatcd noninoculatcd chcck, 2) untrcatcd inoculatcd chcck, 1) 0.21 v:v
Libcrty:watcr, 4) !.!1 v:v Libcrty: watcr, and ) 0.07 molar solution ol urca. Twcnty cars sclcctcd at
random in cach subplot wcrc inoculatcd with a pin bar, using a culturc ol F1V4 A. favus. Fusarium
verticillioides was allowcd to inlcct naturally. Among ycars, inoculatcd cars avcragcd !1.6 mg/Mg to
27.1 mg/Mg morc aatoxin than noninoculatcd cars. Ncithcr Libcrty nor urca rcduccd aatoxin or
lumonisin contamination. Hybrids did not dicr in yicld or aatoxin contamination but onc brand had
lcss lumonisin (1.7 mg/kg and 2.1 mg/kg) than thc othcr (7. mg/kg and 6.9 mg/kg). Grain yiclds wcrc
lcss in 2004 (6.9 Mg/ha) than 200! (8.8 Mg/ha) or 2002 (9.0 Mg/ha).
PANEL DISCUSSION: Crop Management and Handling, Insect Control and
Fungal Relationships
Panel Chair: Pat owd
Panel Members: Patricia Timpcr, ouglas Light, and Arnold 8runs
Panclists dcscribcd various studics. Pat owd indicatcd that thc mycotoxin prcdiction program
dcvclopcd lor Midwcst corn had donc wcll with lumonisins lrom 20002004, and prcdictcd thc
aatoxin which occurrcd in ccntral !llinois in 200. Patty Timpcr indicatcd highcr lcvcls ol aatoxin
whcn ncmatodcs wcrc addcd to thc root zonc, but cccts on pod galling varicd. oug Light rcportcd
that thc pcar cstcr had bccn microcncapsulatcd and whcn combincd with rcduccd ratcs ol dicrcnt
insccticidcs, oltcn providcd signincantly bcttcr ratcs ol control than standard ratcs ol corrcsponding
insccticidcs alonc. Arnold 8runs indicatcd a multiycar study whcrc Libcrty hcrbicidc was applicd to
corn cars at black laycr did not rcsult in any signincant rcductions ol aatoxins.
!n rcsponsc to qucstions, Pat owd indicatcd that thc program was dcsigncd lor Midwcst corn,
but may havc application lor othcr arcas, dcpcnding on how similar hybrids wcrc (no onc lrom
sccd companics providcd lurthcr insight). Hc indicatcd that thc aatoxin prcdictions had not bccn
communicatcd widcly bccausc uppcr ARS managcmcnt was conccrncd about thc limitcd arca thc
program had bccn validatcd and potcntial cccts on thc markct. Hc also indicatcd that a group was
intcrcstcd in hclping with validation ovcr a widcr arca.
!n rcsponsc to qucstions, Patty Timpcr indicatcd that thc root and pod zoncs wcrc watcrcd scparatcly,
so thc pod zonc could rcmain undcr drought whilc thc root zonc could bc watcrcd to kccp thc plants
alivc. Shc also indicatcd that thc mcchanism ol ncmatodc involvcmcnt in tall lcscuc toxic cndophytcs
was not clcar yct.
!n rcsponsc to qucstions, oug Light indicatcd that thc microcncapsulatcd granulcs wcrc ccctivc
lor 21 wccks, and thc twist on attractants wcrc ccctivc lor a month.
Anthocyanins from Petunia Floral Structures that Inhibit Corn Earworm
Development
ric T. Johnson
!
, Patrick F. owd
!
, Mark A. 8crhow
2
!
Crop BioProtection Research Unit, USDA-ARS, National Center for Agricultural Utilization Research,
Peoria IL;
2
New Crops and Processing Research Unit, USDA-ARS, National Center for Agricultural
Utilization Research, Peoria, IL
ur prcvious studics idcntincd a scrics ol anthocyanins othcr than cyanidin (which is lound in corn)
with signincant activity against corn carworms. Vc uscd thrcc dicrcnt lincs ol pctunia that had
altcrnating colorcd and bluc/purplc scctors (subscqucntly lound to bc duc to a combination ol malvidin
and pctunidin glucosidcs) to cxaminc thc cccts ol anthocyanins in an intact plant systcm. Larvac
typically produccd lcss damagc on thc colorcd vs. whitc scctors ol thc pctunia owcrs, and larvac that
lcd on thc colorcd scctors typically wcighcd signincantly lcss that thosc lccding on thc whitc scctors. A
combination ol pctunidin and malvidin glucosidcs uscd in a low protcin dict (to simulatc thc nutritional
contcnt ol thc owcrs) at an approximatc natural conccntration ol !000 and 2000 ppm rcspcctivcly, also
produccd larvac that wcrc signincantly smallcr than thosc lccding on solvcnt control dict.
Ground-Based Remote Sensing for Rapid Selection of Drought and Afatoxin
Resistant Peanut Genotypes
.G. Sullivan
!
and C.C. Holbrook
2
!
USDA-ARS Southeast Watershed Research Laboratory, Tifton, GA;
2
USDA-ARS Crop Genetics and
Breeding Research Unit, Tifton, GA
!n thc Southcastcrn U.S., pcanut produccrs arc challcngcd by long growing scasons and pcriodic drought.
Tc continucd dcvclopmcnt ol drought and aatoxin rcsistant pcanut Arachis hypogaea L.) cultivars is
csscntial to maintain productivity undcr lcss than idcal growing conditions. Rcmotc scnsing ol canopy
rccctancc is a wcllcstablishcd mcthod ol cvaluating crop condition, and thus shows promisc as a ncw
tcchniquc lor thc rapid sclcction ol drought and aatoxin rcsistant pcanut gcnotypcs. Tc objcctivc ol
this study was to cvaluatc ground bascd rccctancc mcasurcmcnts to morc accuratcly quantily small
dicrcnccs in gcnotypc rcsponsc to drought conditions. !n April 2004 scvcral small plots (2 m 2 m)
wcrc cstablishcd at thc Gibbs Farm rcscarch lacilitics in Tilton, GA. Trcatmcnts consistcd nvc pcanut
gcnotypcs cncompassing a rangc ol drought tolcrancc and yicld charactcristics arrangcd in a complctcly
randomizcd block dcsign. rought conditions wcrc simulatcd bcginning 90 days altcr planting and
maintaincd through harvcst. ncc drought conditions wcrc cstablishcd, a handhcld radiomctcr was
uscd to acquirc twicc wcckly rccctancc mcasurcmcnts in thc visiblc and ncar inlrarcd rcgions ol
thc spcctrum. Coincidcnt with rcmotcly scnscd data collcction standard visual ratings and soil watcr
contcnt (0!cm) wcrc acquircd. Scasonal mcasurcmcnts includcd aatoxin and yicld mcasurcmcnts.
ur data indicatc that rcmotcly scnscd data providc morc spccinc and timcly cstimatcs ol gcnotypc
rcsponsc to drought, and could bc uscd to cnhancc brccding progrcss ol drought and aatoxin rcsistant
pcanut varictics.
Correlations Between Biotic Stresses and Afatoxin Contamination in Maize
Matthcw Krakowsky
!
, Xinzhi Ni
!
, and Richard avis
2
!
2
Management Research Unit, Tifton, GA
Aatoxin, a toxin produccd by thc lungus Aspergillus favus, is thc most potcnt carcinogcn lound
in naturc. Aatoxin contamination ol maizc is a chronic problcm in thc southcrn US, whcrc high
tcmpcraturcs, watcr strcss, and inscct damagc producc conditions conducivc to inlcction ol maizc by
A. favus. Tc purposc ol this rcscarch was to dctcrminc thc rclationship bctwccn two biotic strcsscs,
lcal lccding by thc lall armyworm (FAV), Spodoptera frugiperda, and root lccding by thc rootknot
ncmatodc (RKN), Mcloidogync incognita, and contamination ol grain with aatoxin. !n thc nrst
cxpcrimcnt, nvc hybrids (lour commcrcial and onc aatoxin rcsistant) wcrc grown in a splitplot dcsign
with wholc plots rcprcscnting FAV artinciallyinlcstcd or noninlcstcd conditions and splitplots
rcprcscnting a hybrid. FAV damagc was cvaluatcd at scvcn and lourtccn days altcr inlcstation. !n thc
sccond cxpcrimcnt, thrcc commcrcial hybrids wcrc grown in a randomizcd complctcblock dcsign in
a ncld with high population dcnsitics ol RKN. A lumigant ncmaticidc was uscd to crcatc plots with
minimal ncmatodc damagc to comparc to nonlumigatcd plots with a high lcvcl ol ncmatodc damagc.
arly (prcplant), mid, and latc (at harvcst) scason ncmatodc population lcvcls wcrc cstimatcd bascd on
soil samplcs. Corrclations bctwccn plant damagc, plant strcss, and yicld and aatoxin contamination can
bc uscd to cvaluatc thc signincancc ol particular biotic strcsscs on aatoxin contamination ol maizc and
dctcrminc thc locus ol gcnctic improvcmcnt and crop managcmcnt programs.
122
18
TH
SESSION 5: DETECTION, EXTRACTION, AND ANALYSIS OF AFLATOXINS;
POTENTIAL USE OF NATURAL PRODUCTS FOR PREVENTION OF FUNGAL
INVASION AND/OR AFLATOXIN BIOSYNTHESIS IN CROPS
Moderator: Tom Vcdcgacrtncr, Cotton !ncorporatcd
122
Distribution of Afatoxin in Non-irrigated Peanuts
Tomas F. Schatzki and Martin S. ng
Western Regional Research Center, Agricultural Research Service, USDA, Albany, CA
Tc 8 and total aatoxin distribution in Gcorgia orunncr pcanuts has bccn mcasurcd. Samplc
distributions wcrc mcasurcd in approximatcly 400 small samplcs cach ol Jumbo, Mcdium and Small il
Stock (S) sublots, containing 20, !0, and kcrncls, rcspcctivcly. Rcsults wcrc convcrtcd to singlc kcrncl
probability dcnsity (SK) distributions, f(ln c), using mcthods prcviously publishcd ( J Agr Food Chcm
!99, 41: !6!!6). Tis constitutcs thc nrst dircct small samplc cxpcrimcntal cstablishmcnt ol f(ln c)
in pcanuts. All thrcc sublots show cvidcncc in thc SK ol pcaks at about c - !0, !0`, and a partial
pcak at c < !0 ng/g. Tc nrst and last ol thcsc arc similar to pcaks sccn in trcc nuts. Tc cxpcrimcntal
distributions wcrc also nttcd to thc 2paramctcr ncgativc binomial distribution (N8), nrst suggcstcd
lor pcanuts by Vhitakcr and Viscr ( J Amcr il Chcm Soc !969, 46: 177179). Tc N8 lollows
thc cavcragc ol thc cxpcrimcntal data quitc wcll, but docs not rcprcscnt any ol thc pcaks and vallcys.
Tus it can not bc uscd to dcducc inlormation ol nut physiology or growing conditions, which wcrc
so succcsslully dcduccd lor trcc nuts by nonparamctric mcthods. Tc N8 might sumcc to cstimatc
samplc mcan and variancc at samplc sizcs drastically dicrcnt lrom thc samplc sizcs mcasurcd, but this
would rcquirc mcasurcmcnt ol samplcs ol varying sizcs lrom thc samc lot. Such data is prcscntly not
availablc. Comparison ol thc rcsults obtaincd hcrc with thosc obtaincd by Vhitakcr ct al. ( J AAC
!nt !994, 77: 69666) on a largc sct ol similar lots ol Gcorgia orunncrs but with much largcr samplc
sizc (2000 to 6000 pods). Vhcn thc lattcr rcsults wcrc rcduccd by usc ol thc N8 to thc samc sizc as
mcasurcd hcrc, a somcwhat dcgradcd, again pcaklcss, nt was obtaincd. Tis dcgradation was almost
ccrtainly duc to thc samplc variancc which was 10700 largcr in thc Vhitakcr casc. Tcsc rcsults
suggcst, but do not provc, that thc N8 is capablc ol spanning a vcry largc samplc rangc. Prool would
rcquirc largc and small samplcs lrom thc samc lot. 8oth thc prcscnt and thc Vhitakcr data show a cut
o ol contamination occurring slightly abovc !0 ng/g notcd in all trcc nuts prcviously mcasurcd. Tis
is thc nrst tcst ol thc N8 to small samplcs.
Inhibition of Aspergillus favus Afatoxin Biosynthesis by Antioxidant
Phytochemicals Occurring in Tree Nuts
Russcll J. Molyncux, Norccn Mahoncy, 8rucc C. Campbcll and Jong H. Kim
USDA, ARS, Western Regional Research Center, Albany, CA
Valnuts in gcncral and thc cultivar Tularc in particular havc bccn shown to bc cxccptionally rcsistant
to aatoxigcncsis. Tc rcsistancc lactors, locatcd solcly in thc sccd coat or pclliclc but not in thc
kcrncl, havc bccn shown to bc complcx hydrolysablc tannins. Tcsc tannins consist ol a glucosc corc,
cstcrincd by gallic and hcxahydroxydiphcnic acid moictics. Aspergillus favus posscss a tannasc capablc
ol hydrolyzing thc tannins into thcir componcnt parts. Tc phcnolic moitics, gallic acid and cllagic acid
(dcrivcd lrom hcxahydroxydiphcnic acid by spontancous lactonization) also cxhibit antiaatoxigcnic
activity but arc lcss potcnt than thc parcnt tannins. !t has bccn postulatcd that aatoxigcncsis is a
conscqucncc ol oxidativc strcss on thc lungus and that compounds capablc ol rclicving oxidativc strcss
should bc capablc ol rcducing or climinating aatoxin production. Tis hypothcsis has bccn tcstcd by
using Saccharomyces cerevisiae as a modcl lungal systcm to cxaminc lunctional gcnomics ol oxidativc
strcss rcsponscs. Singular gcnc dclction mutants ol S. cerevisiae, cxhibitcd allcviation ol oxidativc strcss
induccd by trcatmcnt with pcroxidc whcn tannic, gallic or cacic acids wcrc prcscnt.
!n ordcr to cxtcnd this nnding to A. favus and to clucidatc structurcactivity rclationships, a numbcr
ol antioxidant phcnolic compounds known to occur in trcc nuts wcrc tcstcd in vitro lor thcir ability to
inhibit aatoxin production in thc prcscncc and abscncc ol pcroxidcinduccd oxidativc strcss. Compounds
tcstcd wcrc: thc hydrolysablc tannins, pcntagalloyl glucosc and 1,digalloyl quinic acid, togcthcr with
quinic acid itscll, thc avonoid, catcchin, and cacic, chlorogcnic, cllagic, 4hydroxybcnzoic and 1,4
dihydroxybcnzoic (protocatcchuic), gallic and vanillic acids. As a modcl lor thc anacardic acids prcscnt
in pistachio hulls, thc commcrcial antioxidant, lauryl gallatc, was also tcstcd.
Biochemical and Genetic Analysis of Gallic Acid in Walnuts in Relation to
Afatoxin Accumulation
Ryann M. Muir
!
, lizabcth !ngham
!
, Sandra Uratsu
!
, Galc McGranahan
!
, Charlcs Lcslic
!
,
Norccn Mahoncy
2
, and Abhaya andckar
!
!
Department of Pomology, University of California, Davis, CA;
2
USDA-ARS, Western Regional Research
Center, Albany, CA
Inhibition of Afatoxin Production by Compounds in Corn Seeds
G.A. Paync
!
, R.A. Holmcs
2
, and R.S. 8oston
2
!
Department of Plant Pathology North Carolina State University, Raleigh, NC;
2
Department of Botany,
North Carolina State University, Raleigh, NC
Vc havc idcntincd compounds prcscnt in kcrncls ol thc rcsistant maizc inbrcd Tcx6 that inucncc
aatoxin (AF) biosynthcsis and lungal growth in bioassays. !n carlicr studics a growth inhibitor was
purincd and dctcrmincd to bc a chitinasc (Moorc et al., Phytopathology 2004, 94: 82 87). Additional
charactcrization ol kcrncl sccd cxtracts has rcvcalcd thc prcscncc ol two nonprotcinaccous compounds
that inucncc AF biosynthcsis and growth: Aatoxin 8iosynthcsis !nhibitor! (A8!!) and A8!2.
A. favus culturcs grown in thc prcscncc ol A8!! cxhibit rcduccd mycclial mat lormation, total
biomass and AF biosynthcsis. !nitial biochcmical charactcrization ol A8!! indicatcs that it is a
nonprotcinaccous, hcat labilc small molcculc. Rclativc to A8!!, A8!2 has lcss inucncc on lungal
growth and a morc pronounccd ccct on AF biosynthcsis. A8!2 is a hcat stablc, nonprotcinaccous
compound. Scvcral lincs ol cvidcncc suggcst that A8!2 bclongs to thc inositol polyphosphatc class ol
molcculcs. Tis is not surprising as maizc sccds storc high lcvcls ol inositol hcxakisphosphatc (phytic
acid) and othcr inositol polyphosphatc prccursors. LC/MS analysis ol A8!2 containing lractions
rcvcalcd cnrichmcnt in inositol polyphosphatcs. Furthcrmorc low phytic acid !! mutant maizc kcrncls
havc rcduccd A8!2 activity and trcatmcnt ol kcrncl cxtracts with phytasc (a phosphatasc) cnhanccs
A8!2 activity. Howcvcr, purc phytic acid had no inhibitory ccct, suggcsting that A8!2 may bc a
biosynthctic prccursor ol phytic acid. Prcliminary rcal timc RTPCR mcasurcmcnts ol AF biosynthctic
and rcgulatory gcnc transcription suggcst that A8!! and A8!2 both supprcss transcription ol pathway
gcncs, but act dicrcntly on othcr rcgulatory gcncs. Tcsc obscrvations show that sccds ol host plants
contain compounds that targct gcnc transcription in A. favus.
PANEL DISCUSSION: Detection, Extraction and Analysis of Afatoxins;
Potential Use of Natural Products for Prevention of Fungal Invasion and/or
Afatoxin Biosynthesis in Crops
Panel Chair: Russcll Molyncux
Panel Members: Gary Paync, Tomas Schatzki
Summary of Panel Discussion: Gary Paync was askcd about thc naturc ol thc two compounds isolatcd
lrom corn sccds that inhibit aatoxin production. Prcvious work had indicatcd that thcsc could bc
protcins. Howcvcr, bascd on mass spcctromctric cvidcncc, thc molccular wcight ol onc was cstimatcd
to bc 69, cvcn though sizc cxclusion chromatography would suggcst a much highcr molccular wcight
compound. Tc oddnumbcrcd molccular wcight would suggcst a nitrogcncontaining compound and
thc chromatographic bchavior could bc cxplaincd by structural lcaturcs that promotc bonding to thc
column matrix.
Russcll Molyncux was askcd about thc rationalc lor antioxidant compounds prcscnt in walnuts to
supprcss aatoxin biosynthcsis. !l aatoxins arc produccd in thc lungus in rcsponsc to oxidativc strcss,
how do thcy protcct thc lungus lrom such strcss: !t was hypothcsizcd that thc luranoid doublc bond
can absorb rcactivc oxygcn spccics in a similar manncr to its mctabolic cpoxidation by P40 cnzymcs
in mammalian systcms. Tc qucstion as to whcthcr or not juglonc could also rclicvc oxidativc strcss
was also poscd. Juglonc itscll, a quinonc, cannot do so bccausc it is lully oxidizcd but it is an artclact
produccd by damagc to walnut hulls, in which it cxists as thc glycosidc ol its rcduccd quinol lorm. Tis
quinol would bc a potcnt antioxidant but cannot protcct thc kcrncls dircctly as it only cxists in thc
hulls. Tcrc was discussion as to whcthcr pistachios and almonds havc antioxidants similar to thosc in
walnuts. Pistachios dcnnitcly havc structurally similar hydrolysablc tannins, although at lowcr lcvcls,
whcrcas almonds do not. Howcvcr, almonds contain condcnscd tannins which may providc somc
dcgrcc ol antioxidant protcction.
Identifcation of Two Maize Seed Compounds that Infuence Afatoxin
Biosynthesis
Robcrt A. Holmcs
!
, Norman J. Glassbrook
2
, Rcbccca S. 8oston
!
, and Gary A. Paync
1
!
Department of Botany, North Carolina State University, Raleigh, NC;
2
School of Biosciences, Cardif
University, Cardif, Wales;
1
Department of Plant Pathology, North Carolina State University, Raleigh, NC
Rcduction ol aatoxin contamination ol maizc and othcr crops would bc lacilitatcd by thc idcntincation
ol host compounds that inucncc sccondary mctabolism in thc aatoxigcnic lungi Aspergillus favus
and A. parasiticus. Vc rcport thc isolation ol two nonprotcinaccous compounds lrom kcrncls ol thc
maizc inbrcd Tcx6 that inhibit aatoxin biosynthcsis in A. favus. Aatoxin 8iosynthcsis !nhibitor!
(A8!!) inhibits growth at high conccntrations but also inucnccs aatoxin biosynthcsis. LCMS
analysis ol A8!! containing lractions has yicldcd scvcral candidatc molcculcs which wc will vcrily
using additional purincation stcps and LCMS. Tc sccond inhibitory compound, A8!2, inhibits
aatoxin biosynthcsis and supprcsscs conidiation, but docs not acct mycclial mass. A8!2 activity docs
not accumulatc in maizc kcrncls until latc in kcrncl dcvclopmcnt (latcr than 28 days altcr pollination).
LCMS analysis ol A8!2 containing lractions shows that inositol hcxakisphosphatc (phytic acid)
is thc major componcnt, with othcr inositol polyphosphatcs prcscnt. Kcrncls lrom low phytic acid -
mutant lincs havc lcss inhibitory activity than wildtypc kcrncls. Trcatmcnt ol inhibitory lractions with
phytasc (a phosphatasc) cnhanccs inhibition ol aatoxin biosynthcsis. Howcvcr, purc phytic acid and
somc othcr inositol polyphosphatc isomcrs havc no strong inhibitory ccct. Likcwisc, trcatmcnt ol purc
phytic acid with phytasc docs not rcsult in inhibition ol aatoxin biosynthcsis. Tus, A8!2 activity
appcars to bc associatcd with thc production ol phytic acid, but not phytatc pcr sc.
A New Peanut Phytoalexin with Stilbene and Tetronic Acid Moieties
\.S. Sobolcv
!
, S.T. cyrup
2
, and J.8. Glocr
2
!
USDA, ARS, National Peanut Research Laboratory, Dawson, GA;
2
e University of Iowa, Department of
Chemistry, Iowa City, IA
A ncw pigmcntcd, low molccular wcight mctabolitc has bccn isolatcd lrom pcanut (Arachis hypogaea)
kcrncls challcngcd by a soil lungal isolatc. Tc structurc ol thc ncw compound, tcrmcd ST!, was
clucidatcd by
!
H and
!1
C NMR, MS, and U\ spcctromctry. Tc ST! molcculc bcars stilbcnc and
tctronic acid moictics and rcprcscnts an unusual class ol compounds. Tc only known ST! analog
was isolatcd lrom hcartwood ol Pericopsis elata. 8oth A. hypogaea and P. elata bclong to thc lamily
Leguminosae. Likc all pcanut stilbcnc phytoalcxins, ST! naturally cxists as thc transisomcr, but can
bc convcrtcd into thc corrcsponding cisisomcr by cxposurc to U\/visiblc light radiation. ST! may bc
rcsponsiblc lor thc ycllow color that is oltcn obscrvcd in highwatcractivity pcanut kcrncls challcngcd
by lungi.
Tc molccular origin ol thc ncw mctabolitc is unknown, howcvcr, thc numbcr ol carbon atoms
corrcsponds to that ol stilbcncs suggcsting that ST! is a dcgradation product ol thc cocxisting stilbcnc,
transArachidin!. Tc orthodihydroxy moicty in Arachidin! might bc oxidativcly dissimilatcd by
bond nssion to producc a ciscismuconic acid dcrivativc. Rcduction ol thc cnol systcm in thc lattcr
dcrivativc to thc dihydromuconic acid dcrivativc lollowcd by lactonization would givc ST!. Although
thc scqucncc in thc abovc schcmc is spcculativc, thc proposcd stcps sccm to bc likcly, bascd on analogs
in thc litcraturc.
Production ol ST! in pcanuts was clicitcd by dicrcnt soil lungi, including toxigcnic and
nontoxigcnic A. favus and A. parasiticus, as wcll as by A. niger and A. caelatus. Tc ncw mctabolitc is
suggcstcd to bc an important rcprcscntativc ol a ncw class ol pcanut phytoalcxins sincc its production
oltcn cxcccds production ol major known stilbcncs. Tc biological activity ol thc ncw compound is thc
subjcct ol luturc invcstigation.

Examination of Error Components Associated with Quantifcation of Afatoxin
in Ground Corn Grain with In-house CD-ELISA
M.J. Clcmcnts
!
, G.L. Vindham
!
, C.M. Maragos
2
, V.P. Villiams
!
, T.. 8rooks
!
, L.K. Hawkins
!
,
and H.M. Gardncr
!
!
USDA-ARS, Corn Host Plant Resistance Research Unit, Mississippi State, MS;
2
USDA-ARS, National
Center for Agricultural Utilization Research, Peoria, IL
Gcnctic rcsistancc is gcncrally considcrcd to bc thc most dcsirablc mcans ol minimizing aatoxin
accumulation in corn grain prior to harvcst, howcvcr, variation associatcd with grain sampling and
subsampling tcchniqucs, and quantitativc analytical protocols grcatly impcdcs accuratc classincation
ol gcnotypcs as rcsistant or susccptiblc to aatoxin accumulation in grain. Somc widcly acccptcd
analytical protocols lor aatoxin quantincation in grain includc liquid chromatography (LC), thinlaycr
chromatography (TLC), immunoamnity column assay (!CA), cnzymclinkcd immunosorbcnt assay
(L!SA), and highpcrlormancc liquid chromatography (HPLC). ach ol thcsc protocols involvcs
scvcral componcnts that may contributc substantial cost or variation to quantincation ol aatoxin
among rcplicatcd analyscs ol thc samc subsamplc. ur objcctivc was to cxaminc crror componcnts and
inputs associatcd with grain sampling and an inhousc compctitivcdircct L!SA (CL!SA) lor
quantincation ol aatoxin in corn grain. Undcrstanding ol crror componcnts associatcd with thc C
L!SA will lcad to managcmcnt dccisions that minimizc cost and maximizc data quality.
Tc CL!SA was dcvclopcd around a monoclonal antibody produccd at thc USAARS
Mycotoxin Rcscarch Unit, Pcoria, !L. Aatoxin conccntration in corn grain dctcrmincd with C
L!SA corrclatcd signincantly (P < 0.000!, r - 0.87) with aatoxin conccntration in grain dctcrmincd
with thc widcly acccptcd \icam AaTcst. Prcliminary analysis ol crror componcnts rcvcalcd that
grcatcst variation in aatoxin conccntration lrom sampling and laboratory assay was cxplaincd by
8gram subsamplcs ol ground grain (6.!), lollowcd by subsubsamplcs ol cxtract solution (24.!), and
subsubsubsampling (!9.8). An incrcasc in subsamplc numbcr lrom ! to 1 dccrcascd LSR ol cntry
mcans by approximatcly 10, whcrcas incrcasing rcplicatcs ol thc assay or incrcasing subsubsamplcs ol
cxtract solution had much lcss ol an ccct on rcducing LSR ol cntry mcans. !ncrcasing subsamplc sizc
lrom 8 to 20, 0, !00, or !0 grams was not bcncncial in rcducing variation in aatoxin conccntration
associatcd with subsampling ol ground grain. Scnsitivity ol thc assay whcn run with thrcc subsamplcs
and a 100lold dilution ol subsamplc cxtract is approximatcly grcatcr than or cqual to 4 ng aatoxin
pcr gram ol ground corn. Tc CL!SA comparcs vcry lavorably with thc widcly acccptcd \icam
AaTcst, thcrclorc thc assay may bc uscd to dicrcntiatc corn gcnotypcs that arc susccptiblc or rcsistant
to aatoxin accumulation in grain.
Using Hyperspectral Technology to Measure Fungal Growth and Assess
Mycotoxin Contamination of Corn
Z. Hruska
!
, H. Yao
!
, K. iCrispino
!
, K. 8rabham
!
, . Lcwis
!
, J. 8cach
!
, R.L. 8rown
2
, and T..
Clcvcland
2
!
Institute for Technology Development, Stennis Space Center, MS;
2
Center, New Orleans, LA
Tc ultimatc aim ol thc currcnt projcct is to dcvclop a rapid, nondcstructivc hypcrspcctral imaging
mcthodology to mcasurc spcctral signaturcs associatcd with lungal inlcction and mycotoxin
contamination ol corn kcrncls. Tc !nstitutc lor Tcchnology cvclopmcnt (!TStcnnis Spacc Ccntcr,
Mississippi) has dcvclopcd a patcntcd, low cost, and portablc tablctop hypcrspcctral imaging systcm
with imaging capability in thc visiblc and ncarinlrarcd rangc (400 and !000 nm). Tc instrumcnt is
idcal lor conducting laboratorybascd cxpcrimcnts, but can also bc adaptcd to ncld applications. !n
prcliminary cxpcrimcnts thc hypcrspcctral scnsor distinguishcd vcry dicrcnt and vcry similar corn
varictics bascd on thcir spcctral signaturcs as wcll as idcntincd uniquc spcctral signaturc lor Aspergillus
favus (A. favus) and dctcrmincd that it is rcadily distinguishablc against any background or surrounding
surlacc and among othcr mold strains.
Tc ncxt logical stcp was to charactcrizc thc A. favus signaturc oncc it inlccts corn and invcstigatc
possiblc changcs in thc spcctral pattcrn ol A. favus on corn acctcd by daily growth. !t was also
important to comparc lungal growth with an cstablishcd chcmical assay such as thc kcrnclscrccning
assay in ordcr to dctcrminc il mycotoxin contamination on corn corrclatcs with thc spcctral pattcrn ol
A. favus. Tcrclorc, thc objcctivc ol thc prcscnt cxpcrimcnt was to obscrvc any changcs in thc spcctral
signaturc ol A. favus on corn kcrncls ovcr an cightday growth pcriod. !n addition, thc daily aatoxin
production was mcasurcd and corrclatcd with any obscrvcd changc in thc spcctral signaturc.
A corn linc that has bccn lound to consistcntly producc a robust toxin rcsponsc was uscd lor aatoxin
inlcction. Corn kcrncls wcrc inoculatcd with A. favus and placcd into a 10 C/!00 humidity incubator
lor 8 days. Tc kcrncls wcrc inlcctcd in an inoculum madc lrom A. favus (AF!1) culturcs at a dilution ol
4 !0 sporcs/ml. Fivc dishcs (! control and 4 trcatmcnt rcps) cach containing 4 kcrncls, wcrc imagcd
cach day to cstablish a daily A. favus growth pattcrn on corn. \N!R imaging bcgan on thc nrst day ol
growth, 24 hours altcr inoculation. Following \N!R imaging, kcrncls lrom cach day wcrc placcd into
a 60 C ovcn lor 2 days to tcrminatc lurthcr mold growth in prcparation lor thc kcrncl scrccning assay
(KSA). ry kcrncls wcrc proccsscd lor thc KSA according to thc protocol dcvclopcd at SRRC. Tc
imaging data wcrc proccsscd and analyzcd using imagc analysis mcthods and algorithms dcvclopcd lor
corn at !T.
Tc rcsults suggcst that thcrc is a signincant spcctral signaturc changc bctwccn thc growth on ay !
and thc growth on thc rcmaining days throughout thc spcctra (40 900 nm). A signincant signaturc
dicrcncc bctwccn ay 2 and thc rcmaining days cxists lrom 40 to 78! nm. Tc rcsults also rcvcalcd
a corrclation bctwccn toxin lcvcl and avcragc rccctancc at spccinc bands throughout thc spcctra.
stablishing corrclation bctwccn hypcrspcctral data and thc chcmical kcrnclscrccning assay ovcr a
growth pcriod is thc nrst stcp toward quantilying toxin on corn kcrncls.
132 P~v:iciv~x:s
P~v:iciv~x:s 133
Participants
Abbas, Hamed K.
USDA, ARS
Crop Genetics & Production Research Unit
P.O. Box 345
Stoneville, MS 38776-0345
662-686-5313
habbas@msa-stoneville.ars.usda.gov
Antilla, Larry C.
Arizona Cotton Research & Protection Council
3721 East Wier Ave.
Phoenix, AZ 85040-2933
602-438-0059
lantilla@azcotton.org
Appell, Michael
USDA, ARS, NCAUR
Mycotoxin Research Unit
1815 N. University
Peoria, IL 61604
309-681-6249
appellm@ncaur.usda.gov
Averhoff, Scott
Texas Corn Producers Board
P.O. Box 999
Waxahachie, TX 75168
972-937-2587
scottaverhoff@hotmail.com
Balint-Kurti, Peter J.
USDA, ARS
Plant Science Research Unit
NC State Dept. of Plant Pathology, Gardner Hall
Raleigh, NC 27695-7616
919-515-3516
peter_balintkurti@ncsu.edu
Bertels, Paul J.
National Corn Growers
632 Cepi Drive
Chesterfield, MO 63005
636-733-9004
bertels@ncga.com
Betrn, Javier
Texas A&M University
Soil and Crop Sciences Dept.
Heep Building TAMU
College Station, TX 77843-2474
979-845-3469
javier-betran@tamu.edu
Bridges, Susan M.
Mississippi State University
Dept. of Computer Science & Engineering
Box 9637
Mississippi State, MS 39762
662-325-7505
bridges@cse.msstate.edu
Brooks, Thomas D.
USDA, ARS
Corn Host Plant Resistance Research Unit
345 Dorman Hall, Stone Blvd.
662-325-7996
tbrooks@msa-msstate.ars.usda.gov
Brown, Robert L.
USDA, ARS, SRRC
Food and Feed Safety Research Unit
1100 Robert E. Lee Blvd.
New Orleans, LA 70124
225-578-1216
rbrown@srrc.ars.usda.gov
Brown, Daren
USDA, ARS, NCAUR
1815 N. University
Peoria, IL 61604
309-681-6230
browndw@ncaur.usda.gov
Bruns, H. Arnold
USDA, ARS
Crop Genetics & Production Research Unit
P.O. Box 345
Stoneville, MS 38776-0345
662-686-5279
abruns@msa-stoneville.ars.usda.gov
Butchko, Robert
USDA, ARS, NCAUR
1815 N. University
Peoria, IL 61604
309-681-6073
butchkora@ncaur.usda.gov
Calvo-Byrd, Ana M.
Northern Illinois University
Dept. of Biological Sciences
1425 W Lincoln Hwy
Dekalb, IL 60115
815-753-0451
amcalvo@niu.edu
132 P~v:iciv~x:s
P~v:iciv~x:s 133
Camas, Alberto
Dept. of Biochemistry & Molecular Biology
Dorman Hall, Stone Blvd.
662-325-7747
ac201@msstate.edu
Campbell, Bruce C.
USDA, ARS, WRRC
Plant Mycotoxin Research Unit
800 Buchanan St
Albany, CA 94710-1100
510-559-5846
bcc@pw.usda.gov
Cantrell, Roy G.
Cotton Incorporated
6399 Weston Parkway
Cary, NC 27513
919-678-2266
rcantrell@cottoninc.com
Carbone, Ignazio
NC State University
Center for Integrated Fungal Research
Dept. of Plant Pathology
919-513-4866
ignazio_carbone@ncsu.edu
Cary, Jeff
USDA, ARS, SRRC
504-286-4264
jcary@srrc.ars.usda.gov
Chen, Zhi-Yuan
Dept. of Plant Pathology & Crop Physiology
LSU Agricultural Center
Baton Rouge, LA 70803
225-578-1464
zchen@agcenter.lsu.edu
Clements, Michael J.
USDA, ARS
Box 955
662-325-8023
mclements@msa-msstate.ars.usda.gov
Cotty, Peter J.
USDA, ARS, SRRC
Div. of Plant Pathology & Microbiology
University of Arizona
1140 E. South Campus Drive
Tucson, AZ 85721-0036
520-626-5049
pjcotty@email.arizona.edu
Czika, Carol A.
Cotton Incorporated
6399 Weston Parkway
Cary, NC 27513
919-678-2270
cczika@cottoninc.com
Damann, Kenneth E.
Dept. of Plant Pathology & Crop Physiology
LSU Agricultural Center
Baton Rouge, LA 70803
225-578-1401
kdamann@agctr.lsu.edu
Davis, Georgia L.
University of Missouri-Columbia
Division of Plant Sciences
1-31 Agriculture
Columbia, MO 65211
573-882-9224
davisge@missouri.edu
Dorner, Joe W.
USDA, ARS
National Peanut Research Laboratory
P.O. Box 509, 1011 Forrester Dr. SE
Dawson, GA 39842
229-995-7408
jdorner@nprl.usda.gov
Doster, Mark A.
University of California
Kearney Agricultural Center
9240 S. Riverbend Ave.
Parlier, CA 93648
559-646-6598
mark@uckac.edu
Dowd, Patrick F.
USDA, ARS, NCAUR
Crop Bioprotection Research Unit
1815 N. University St.
Peoria, IL 61604
309-681-6242
dowdpf@ncaur.usda.gov
134 P~v:iciv~x:s
P~v:iciv~x:s 135
Eubanks, Mary W.
Duke University
Department of Biology
Durham, NC 27708-0338
919-660-7419
eubanks@duke.edu
Felton, Gary W.
Penn State University
Dept. of Entomology
501 ASI Bldg.
University Park, PA 16804
814-863-7789
gwf10@psu.edu
Georgianna, D. Ryan
NC State University
Center for Integrated Fungal Research and
Department of Plant Pathology
Box 7567
919-515-6995
drgeorgi@ncsu.edu
Gibson, David
Texas Corn Producers Board
4205 N. I-27
Lubbock, TX 79403
806-763-2676
dgibson@texascorn.org
Glassbrook, Norm
NC State University
851 Main Campus Drive
267 Partners III
Raleigh, NC 27606
919-451-5060
njglassb@ncsu.edu
Godshalk, Brent
BASF Plant Science
26 Davis Drive
Research Triangle Park, NC 27709
919-547-2883
godshae@basf-corp.com
Gorman, Daniel P.
Pioneer Hi-Bred
2300 Industrial Park Rd. NE
Cairo, GA 39828
229-378-8240
dan.gorman@pioneer.com
Gradziel, Tom
Plant Sciences
Davis, CA 95616
530-752-1575
tmgradziel@ucdavis.edu
Guo, Baozhu
USDA, ARS
Crop Protection & Management Research Unit
P.O. Box 748
Tifton, GA 31793-0748
229-387-2334
bguo@tifton.usda.gov
Hammond, Bruce G.
Monsanto Co.
800 North Lindbergh Boulevard
Mail Zone: O3C
St. Louis, MO 63167-0001
314-694-8482
bruce.g.hammond@monsanto.com
Hawkins, Leigh K.
USDA, ARS
662-325-2707
lhawkins@msa-msstate.ars.usda.gov
Highland, Brett
AgraQuest, Inc
1069 Eisenhower Dr
Nokomis, FL 34275
941-484-4523
bhighland@agraquest.com
Holbrook, Corley
USDA, ARS
Crop Genetics and Breeding Research Unit
P.O. Box 748
Tifton, GA 31793-0748
229-386-3176
holbrook@tifton.usda.gov
Holland, James B.
USDA, ARS
Plant Science Research Unit
Dept. of Crop Science, Box 7620 NC State
919-513-4198
james_holland@ncsu.edu
134 P~v:iciv~x:s
P~v:iciv~x:s 135
Holmes, Robert A.
NC State University
Dept. of Botany, Campus Box 7612
919-515-6995
raholme2@unity.ncsu.edu
Horn, Bruce
USDA, ARS
Dawson, GA 39842-0509
229-995-7410
bhorn@nprl.usda.gov
Hruska, Zuzana
Institute for Technology Development
Building 1103, Suite 118
Stennis Space Center, MS 39529
228-688-2509
zhruska@iftd.org
Hua, Sui Sheng
USDA, ARS, WRRC
800 Buchanan St
Albany, CA 94710
510-559-5905
ssth@pw.usda.gov
Isleib, Thomas G.
NC State University
Dept. of Crop Science, Box 7629
919-515-3281
tom_isleib@ncsu.edu
Jacobs, Merle
Almond Board of California
1150 9th Street Suite 1500
Modesto, CA 95354
209-343-3222
mjacobs@almondboard.com
Jacobus, Carrie A.
NC State University
851 Main Campus Dr.
267 Partners III
Raleigh, NC 27606
919-515-6995
cajacobu@unity.ncsu.edu
Jaime-Garcia, Ramon
Div. of Plant Pathology & Microbiology
P. O. Box 210036-0036
Tucson, AZ 85721
520-626-7855
rjaime@email.arizona.edu
Jaramillo, Frank
Office of the Texas State Chemist
Reed McDonald Building Room 302
College Station, TX 77836
979-845-1121
Jones, Don C.
Cotton Incorporated
6399 Weston Parkway
Cary, NC 27513
919-678-2367
djones@cottoninc.com
Kendra, David
USDA, ARS, NCAUR
1815 N. University
Peoria, IL 61604
309-681-6579
kendrad@ncaur.usda.gov
Kerns, Mike
Monsanto Co.
3302 SE Convenience Blvd.
Ankeny, IA 50021
515-963-0621
mike.kerns@monsanto.com
Keys, Lynda S.
Cotton Incorporated
6399 Weston Parkway
Cary, NC 27513
919-678-2269
lkeys@cottoninc.com
Kolomiets, Mike V.
Dept. of Plant Pathology and Microbiology
2132 TAMU
979-458-4624
kolomiets@tamu.edu
136 P~v:iciv~x:s
P~v:iciv~x:s 137
Krakowsky, Matthew
USDA, ARS
Crop Genetics and Breeding Research Unit
P.O. Box 748
Tifton, GA 31793-0748
229-387-2341
mkrakowsky@tifton.usda.gov
Lee, Robert Dewey
University of Georgia
15 RDC Road RDC-CES-UGA
Tifton, GA 31793
229-386-3432
deweylee@uga.edu
Light, Douglas M.
USDA, ARS, WRRC
800 Buchanan Street
Albany, CA 94710
530-304-9302
dlight@pw.usda.gov
Luo, Meng
2747 David Rd.
Tifton, GA 31794
229-387-2377
mluo@tifton.uga.edu
Luthe, Dawn
MS State University
Dept. of Biochemistry, Box 9650
662-325-7733
dsluthe@ra.msstate.edu
Mahoney, Noreen
USDA, ARS, WRRC
800 Buchanan Street
Albany, CA 94710
510-559-5971
nmahoney@pw.usda.gov
Mayfield, Kerry L.
Soil & Crop Sciences Dept., MS 2474
979-845-3469
kerry-mayfield@tamu.edu
Milla, Susana R.
NC State University
919-515-3196
susana_milla@ncsu.edu
Molyneux, Russell J.
USDA, ARS, WRRC
800 Buchanan Street
Albany, CA 94710
510-559-5812
molyneux@pw.usda.gov
Moore, Steven
Dean Lee Research Station
8105 Tom Bowman Drive
Alexandria, LA 71302
318-473-6524
smoore@agcenter.lsu.edu
Muir, Ryann M.
Dept. of Pomology, One Shields Ave.
Davis, CA 95616
530-752-5325
ryannmuir@hotmail.com
Murphy, Emory M.
Georgia Peanut Commission
P.O. Box 967
Tifton, GA 31793-0967
229-386-3470
emory@gapeanuts.com
Nichols, Robert L.
Cotton Incorporated
6399 Weston Parkway
Cary, NC 27513
919-678-2371
bnichols@cottoninc.com
Nielsen, Ray F.
Smart World Organics, Inc.
18744 Titus Road
Hudson, FL 34667
727-697-3661
ray@smartwp.com
136 P~v:iciv~x:s
P~v:iciv~x:s 137
Nierman, William
TIGR
9712 Medical Center Drive
Rockville, MD 20850
301-424-4316
wnierman@tigr.org
Nwosu, Victor
Masterfoods, USA
800 High St.
Hackettstown, NJ 7840
908-850-7545
victor.nwosu@effem.com
OBrian, Greg
NC State University
Dept. of Plant Pathology
851 Main Campus Drive
Raleigh, NC 27695
919-515-6995
greg_obrian@ncsu.edu
Olsen, Mary W.
Dept. of Plant Sciences
1140 E. South Campus Dr. Forbes 204
Tucson, AZ 85721
520-626-2681
molsen@ag.arizona.edu
Ozias-Akins, Peggy
Department of Horticulture and NESPAL
P.O. Box 748
Tifton, GA 31793-0748
229-386-3902
pozias@uga.edu
Palumbo, Jeffrey
USDA, ARS, WRRC
800 Buchanan St.
Albany, CA 94710
510-559-5876
palumbo@pw.usda.gov
Payne, Gary
NC State University
Department of Plant Pathology, Box 7567
223 Partners III
Raleigh, NC 27695
919-515-6994
gary_payne@ncsu.edu
Peethambaran, Bela
21H Wallace Circle
Starkville, MS 39759-3084
662-341-1293
bp64@msstate.edu
Perkins, James M.
Monsanto Co.
8350 Minnegan Rd.
Waterman, IL 60556-7113
815-758-9524
jim.perkins@monsanto.com
Pritchard, Bethan
NC State University
2124 Gorman St.
Raleigh, NC 27606
919-247-2901
beth_pritchard@ncsu.edu
Proctor, Robert
USDA, ARS, NCAUR
1815 N. University
Peoria, IL 61604
309-681-6380
proctorh@ncaur.usda.gov
Raab, Quinton J.
B-H Genetics
300 S Lancaster Street PO Box 620
Moulton, TX 77975-0620
361-596-4088
qraab@sbcglobal.net
Rajasekaran, Kanniah
USDA, ARS, SRRC
504-286-4482
krajah@srrc.ars.usda.gov
Rathore, Keerti S.
Inst. For Plant Genomics & Biotechnology
2123 TAMU Borlaug Center
979-862-4795
rathore@tamu.edu
138 P~v:iciv~x:s
P~v:iciv~x:s 139
Richard, John
Romer Labs, Inc.
1301 Stylemaster Dr.
Union, MO 63084
636-583-8600
johnr@romerlabs.com
Robens, Jane F.
USDA, ARS
National Program Leader Food Safety
Rm 4-2184 GWCC
5601 Sunnyside Avenue
Beltsville, MD 20705-5138
301-504-5381
jfr@ars.usda.gov
Robertson, Leilani A.
NC State University
Dept. of Crop Science
2122 Williams Hall, Box 7620
Raleigh, NC 27695
919-515-4087
larobert@unity.ncsu.edu
Schatzki, Thomas F.
USDA, ARS, WRRC
800 Buchanan St.
Albany, CA 94710
510-559-5672
tom@pw.usda.gov
Shim, Won-Bo
Dept. of Plant Pathology & Microbiology
2132 TAMU
979-458-2190
wbshim@tamu.edu
Simmons, Mary W.
USDA, ARS
National Program Leader Crop Production and
Protection, Rm 4-2210 GWCC
5601 Sunnyside Ave.
Beltsville, MD 20705
301-504-5560
kws@ars.usda.gov
Smith, David R.
Zea Sage
1834 West Forestview Drive
Sycamore, IL 60178
815-501-1659
dsmith1834@tbcnet.com
Sobolev, Victor
USDA, ARS
Dawson, GA 39842
229-995-7446
vsobolev@nprl.usda.gov
Starr, Magen R.
NC State University
Raleigh, NC 27695
217-377-6883
mrstarr@ncsu.edu
Sullivan, Dana
USDA, ARS
Southeast Watershed Research Laboratory
P.O. Box 748
Tifton, GA 31793-0748
229-386-3665
dgs@tifton.usda.gov
Timper, Patricia
USDA, ARS
P.O. Box 748
Tifton, GA 31793
229-386-3188
ptimper@tifton.usda.gov
Tonos, Jennifer L.
USDA, ARS
Crop Genetics and Production Research Unit
NBCL, 59 Lee Rd. Building B
Stoneville, MS 38776
662-686-5230
jtonos@msa-stoneville.ars.usda.gov
Valentine, Howard E.
American Peanut Council
10625 Big Canoe
Jasper, GA 30143
706-579-1755
pnuttech@alltel.net
Voss, Kenneth A.
USDA, ARS, RBRRC
Toxicology and Mycotoxin Research Unit
950 College Station Road
Athens, GA 30605
706-546-3315
kvoss@saa.ars.usda.gov
138 P~v:iciv~x:s
P~v:iciv~x:s 139
Wakelyn, Phillip J.
National Cotton Council
1521 New Hampshire
Washington, DC 20036
202-745-7805
pwakelyn@cotton.org
Walker, Scott
Monsanto Co.
9631 Hedden Road
Evansville, IN 47725
812-867-7183
scott.l.walker@monsanto.com
Wang, Xingfen
NC State University
Raleigh, NC 27695
919-515-2704
xwang15@unity.ncsu.edu
Wedegaertner, Tom C.
Cotton Incorporated
6399 Weston Parkway
Cary, NC 27513
919-678-2369
twedegaertner@cottoninc.com
Weissinger, Arthur K.
NC State University
Dept. of Crop Science, Box 7620 NCSU
919-515-2704
arthur@unity.ncsu.edu
White, Don G.
University of Illinois
Crop Sciences Dept., N425 Turner Hall
1102 South Goodwin Ave.
Urbana, IL 61801
217-333-1093
donwhite@uiuc.edu
Wicklow, Donald
USDA, ARS, NCAUR
1815 N. University
Peoria, IL 61604
309-681-6243
wicklodt@ncaur.usda.gov
Willcox, Martha
2501 Porter St. NW #710
Washington, DC 20008
202-248-2232
mwillcox@earthlink.net
Williams, W. Paul
USDA, ARS
662-325-2735
wpwilliams@msa-msstate.ars.usda.gov
Windham, Gary L.
USDA, ARS
662-325-2594
glwindham@msa-msstate.ars.usda.gov
Wisser, Randall J.
Cornell University
Institute for Genomic Diversity
160 Biotechnology Bldg.
Ithaca, NY 14853
607-255-0761
rjw29@cornell.edu
Woloshuk, Charles P.
Purdue University
Dept. of Botany and Plant Pathology
915 W. State Street
West Lafayette, IN 47907-2054
765-494-3450
woloshuk@purdue.edu
Wra, Lawrence J.
BASF Plant Science
26 Davis Drive
Research Triangle Park, NC 27709
919-547-2004
neal@basf.com
Wu, Minsheng
NC State University
Raleigh, NC 27695
919-515-2704
minsheng_wu@ncsu.edu
Wynne, Johnny C.
NC State University
Deans Office
College of Ag and Life Sciences
Raleigh, NC 27695
919-515-2668
johnny_wynne@ncsu.edu
140 P~v:iciv~x:s
Au:nov !xuvx 141
Xu, Wenwei
Agricultural Research and Extension Center
Route 3, Box 219
Lubbock, TX 79403
806-746-4015
we-xu@tamu.edu
Yao, Haibo
Institute for Technology Development
976 Maple Creek Dr.
Slidell, LA 70461
228-688-2509
hyao@iftd.org
Yu, Jiujiang
USDA, ARS, SRRC
301-795-7570
jiuyu@srrc.ars.usda.gov
Zhang, Jinfen
Agricultural Research and Extension Center
Route 3, Box 219
Lubbock, TX 79403
806-746-6101
TSNYJ999@yahoo.com.cn
Zimeri, Anne Marie S.
USDA, ARS, RBRRC
Toxicology & Mycotoxin Research Unit
950 College Station Road
Athens, GA 30605
706-546-3188
azimeri@saa.ars.usda.gov
140 P~v:iciv~x:s
Au:nov !xuvx 141
Author Index
A
Abbas, H. K. 77, 81, 117
Andcrson, A. 99
Antilla, L. 93
Appcll, M. 59
Arnold, K. 81
B
8acon, C. V. 62
8akcr, J. L. 97
8alintKurti, P. 57
8aushcr, M. 70
8cach, J. 131
8crhow, M. A. 119
8ctrn, J. 66, 67, 80, 81, 82, 83
8hatnagar, . 35, 36, 37, 39, 45
8luhm, 8. 51, 60
8occklcr, L. 95, 96
8oston, R. S. 126, 128
8ouyssounousc, P. 116
8oykin, . L. 44
8rabham, K. 131
8ridgcs, S. 72, 75
8rooks, T. 71, 81, 130
8rown, . V. 38, 58, 60
8rown, R. L. 78, 110, 131
8runs, H. A. 117
8urns, T. . 63
8ush, . 89
8usman, M. 38, 58, 60
8utchko, R. A. . 38, 58, 60
C
Calvo, A. M. 40
Camas, A. 84
Campbcll, 8. C. 35, 37, 45, 116, 124
Cantonwinc, . 76
Carbonc, !. 43
Cary, J. V. 40, 107, 111
Chan, K. L. 35, 45
Chcn, H. 70
Chcn, Z.Y. 78, 110
Chu, Y. 107
Clcmcnts, M. J. 81, 88, 130
Clcvcland, T. . 35, 36, 37, 39, 45, 78, 110, 111, 131
Cotty, P. J. 73, 93, 94, 95, 96, 102, 103, 104
Cox, J. . 43
Coy, A. . 70
Culbrcath, A. 70
D
amann, K. . 105, 110
andckar, A. M. 69, 125
ang, P. 70
avis, . 70, 86, 89, 121
avis, G. 89
can, R. 39
csjardins, A. . 58
cyrup, S. T. 129
iCrispino, K. 131
orncr, J. V. 92
ostcr, M. 95, 96
owd, P. F. 114, 119
oylc, J. 96
uran, R. M. 40
E
dwards, M. 83
F
Faustinclli, P. 107
Fcdorova, N. . 36
Fclts, . 95, 96
Fcrguson, L. 96
Fcussncr, !. 54
Fucntcs, M. 61
G
Gao, X. 54
Garbcr, N. P. 104
Gardncr, H.M. 130
Gclincauvan Vacs, J. 8. 63
Gcorgianna, . R. 47
Gcrau, M. 89
Glassbrook, N. J. 49, 128
Glcnn, A. . 55, 61
Glocr, J. 8. 129
Gorman, . 81
Gradzicl, T. M. 69
Guo, 8. Z. 70, 76, 81, 86, 87
H
Hawkins, L. K. 44, 72, 109, 130
Hinton, . M. 62
Hodgcs, J. . 75
Holbrook, C. C. 70, 76, 87, 107, 115, 120
Holland, J. 8. 52, 57, 85
Holmcs, R.A. 126, 128
Horn, 8. V. 43, 91
Hruska, Z. 131
Hua, S.S. 98
142 Au:nov !xuvx
Au:nov !xuvx 143
Husman, S. 73
Hutmachcr, 8. 111
I
!ngham, . 125
!sakcit, T. 66, 67, 80, 81, 83
J
Jacobus, C. 48
Jaimc, R. 102
Jakobck, J. L. 43
Jayncs, J. M. 111
Jccrs, . 82
Jha, A. 105
Jincs, M. P. 57
Johnson, . T. 119
K
Kcllcy, R. Y. 44
Kcndra, . F. 59, 68, 99
Kim, H. S. 37
Kim, J. H. 35, 45, 124
Kolomicts, M. 54
Krakowsky, M. . 70, 71, 81, 82, 121
Kvicn, C. K. 70, 76
L
Lcc, R. . 70, 86
Lcslic, C. 125
Lcwis, . 131
Liang, X 70, 87
Liang, X. 87
Light, . M. 116
Lopcz, L. 84
Lopcz dc Pratdcsaba, L. 61
Luo, M. 70, 86
Luthc, . S. 72, 75, 84, 109
M
Magcc, G. 8. 75
Mahoncy, N. . 45, 97, 124, 125
Maragos, C. M. 59, 130
Mashida, M. 36
May, G. S. 35, 45
Mayncld, K. 66, 67, 80
McAlpin, C. . 99
McGranahan, G. 125
McKcc, C. 80
Mcnkir, A. 78
Mcnz, M. 83
Michailidcs, T. 95, 96
Millard, M. 77
Molyncux, R. J. 45, 124
Moorc, S. 77, 81
Morgan, . 95, 96
Morriss, C. 89
Moussa, . H. 43
Muhitch, M. J. 99
Muir, R. M. 125
N
Ni, X. 121
Nicrman, V. C. 36, 37, 39
Njapau, H. 103
O
onncll, K. 61
dvody, G. 66, 67, 74, 80
lscn, M. V. 73
ng, M. S. 123
ziasAkins, P. 70, 107
P
Palcncia, . 61
Palumbo, J. . 97
Paync, G. A. 35, 36, 37, 39, 45, 47, 48, 49, 57, 85,
126, 128
Pcarson, T. C. 53
Pcchan, T. 72
Pcchanova, . 72
Pccthambaran, 8. 72, 109
Pcrkins, J. 81
Plattncr, R. . 58
Pricc, M. S. 47
Pritchard, 8. 39
Probst, C. 103
Proctor, R. H. 38, 58, 60
R
Raab, Q. 81
Rajasckaran, K. 107, 111
Ramos, L. 107
Rcycs, H. 95, 96
Rcynolds, K. M. 116
Rilcy, R. T. 55, 61, 63
Robcrtson, L. A. 57, 85
Robcrtson, N. 48
Rochclord, T. 65
Ronning, C. M. 36
S
Sagaram, U. S. 46
Schatzki, T. F. 123
Shim, V.8. 46, 54
142 Au:nov !xuvx
Au:nov !xuvx 143
Smith, . 81
Sobolcv, \. S. 129
Starr, M. 85
Sullivan, . 76, 120
Swcany, R. 105
T
Timpcr, P. 76, 115
Torrcs, . A. 61
U
Ulloa, M. 111
Uratsu, S. 125
V
\oss, K. A. 63
W
Vang, N. 75
Vcissingcr, A. K. 108
Vhitc, . G. 57, 65, 81
Vicklow, . T. 53
Vilkinson, J. R. 37
Villiams, L. . 55
Villiams, V. P. 44, 71, 72, 74, 75, 81, 82, 84, 88,
109, 130
Vilson, . M. 76, 115
Vindham, G. L. 71, 72, 81, 109, 130
Voloshuk, C. 51, 60
Vortman, J. 36
X
Xu, V. 70, 74, 81, 86
Y
Yao, H. 131
Yu, J. 35, 36, 37, 39, 45
Z
Zhang, J. 74
Zimcri, A. 55

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Proceedings of the 2005 Annual

, thc biocontrol product

' lor AF117, 121 ng

' lor F!, 206 ng g

' lor L!, and 06 ng g

' lor !. !n hybrids, aatoxin avcragcs wcrc !29 ng g

' lor F!, !18 ng g

' lor L!, and !97 ng g

' lor !. !solatcs F! and ! produccd morc

University of Columbia, MO;

' at Collcgc Station, 86 ng g

' at Corpus Christi, !1! ng g

' at Vharton, 407 ng g

' at 8ardwcll, and 274 ng g

' at Prospcr. vcrall, wc lound that thc commcrcial

'. Aatoxin conccntration was positivcly

Texas A&M University, Lubbock, TX;

' at Tilton, 192 ng g

' at Starkvillc, !82 ng

' at Urbana and 62 ng g

' at Vcslaco. Signincant G! was obscrvcd lor both

' at Ganado, and !14 ng g

' at Vcslaco. Aatoxin conccntrations lor thc rcst ol thc

USDA-ARS, Tifton, GA;

' lor whitc inbrcds and 4! ng g

' in Vcslaco, TX. Ycllow hybrids CML4! CL02844 and CL

') than Vcslaco (2!4 ng g

'). vcrall mcan lor thc population across locations was

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