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Sensitivity of Wheat Genotypes to a Toxic Fraction Produced Bycephalosporium

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Sensitivity of Wheat Genotypes to a Toxic Fraction Produced Bycephalosporium AN ABSTRACT OF THE THESIS OF Mahfuzur Rahman for the degree of Master of Science in Botany and PlantPathology presented on September 19, 2000. Title: Sensitivity of Wheat Genotypes to a Toxic Fraction Produced byCephalosporium ramineum and Correlation with DiseaseSusceptibility Abstract approved: Redacted for Privacy Christopher C. Mundt Thorhas J. Wolpert Cephalosporium stripe, caused by the soil-borne ascomycete Cephalosporium gramineum, is becoming an increasingly important disease of winter wheat(Triticum aestivum) in several areas of the world, especially where stubble mulch is practiced to maintain soil moisture and prevent erosion. As cultural control of the disease is infeasible and no fungicides are registered, the development of resistant cultivarsoffers the best hope for disease control. Selection of resistant genotypes remainsproblematic due to the requirements of evaluating adult plants in variable field environments.The symptoms of cephalosporium stripe suggest the involvement ofpathogen-produced toxins, and the toxin called graminin A has previously been isolated from C. gramineum. The goals of this thesis were to determine if insensitivity of wheat genotypes to a toxic fraction produced by C. gramineum is associatedwith resistance to cephalosporium stripe, and to evaluate the potential use of this toxic fraction to screen wheat genotypes for disease resistance. A method was developed to mass-produce a toxic fraction of C. gramineum by modifying the method of K. Kobayashi (Kobayashi and Ui 1977). Large volumes (9 L) of broth medium were inoculated with C. gramineum and incubated for 35 days. The culture filtrate was then extracted four times with ethyl acetate, which eliminated need for the most time-consuming step of rotary evaporation. Reversed phase fractionation of the crude extract was used to isolate the toxic fraction. Leaves from fourteen-day-old wheat plants were excised and placed in scintillation vials containing the toxic fraction. Exposure to a concentration of(60d1ml) of the toxic fraction for 72 hours produced distinct wilting symptoms that allowed us to distinguish wheat genotypes in a repeatable manner. Degree of wilting was measured on a continuous 1-5 scale, where 1 = no wilting and 5 = a filly wilted leaf. Twenty wheat genotypes, belonging to four distinct germplasm groups (common, club, durum, and synthetic) were included in three runs of the toxin assay. Wilting reactions for the 20 genotypes were very consistent among runs. Sensitivity of the individual genotypes to the toxic fraction did not differ significantly within germplasm groups, but all differences among the means of the four germplasm groups were highly significant (P < 0.001) based on linear contrasts. Seventeen winter wheat genotypes representing the common, club, and durum germplasm groups were planted in infested fields at two locations. The percentage of tillers showing whitehead symptoms (early maturity and reduced grainfill) was significantly correlated with wilting symptoms measured by the toxin assay. The common and durum genotypes varied more for disease susceptibility than for toxin insensitivity. However, all pairwise linear contrasts between means of the whitehead percentages for the three germplasm groups were highly significant (P < 0.001). Failure of the toxin assay to identify some of the moderately resistant common wheat genotypes suggests that additional mechanisms of resistance are operative. As no toxin-insensitive genotype showed susceptibility to cephalosporium stripe in the field experiments, however, the toxin assay can be used as an initial screening procedure to reduce the number of genotypes to be tested in the field. Copyright by Mahfuzur Rahman September 19, 2000 All Rights Reserved Sensitivity of Wheat Genotypes to a Toxic Fraction Produced by Cephalosporium gramineum and Correlation with Disease Susceptibility by Mahfuzur Rahman A THESIS Submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented September 19, 2000 Commencement June 2001 Master of Science thesis of Mahfuzur Rahman presented on September 19, 2000 Approved: Redacted for Privacy Co-Major Professor, representing Botany and Plant Pathology Redacted for Privacy Botany and Plant Pathology Redacted for Privacy Char of Department of Botanythd Plant pathology Redacted for Privacy Dean of I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for Privacy Author ACKNOWLEDGEMENTS I would like to thank the members of my committee, Christopher C. Mundt, Thomas J. Wolpert, and Oscar RieraLizarazu for their advice, support, and encouragement during this research. I am highly indebted to Christopher C. Mundt and Thomas J. Wolpert, my major professors, for shaping and guiding the research at every step. This specific project would not have been possible without the clear insight of Thomas J. Wolpert about toxin activity and practical assistance in extracting the toxic fraction in a simplified method for mass production. Christopher C. Mundt made it possible for me to conduct experiments in farmers' fields, which I hereby gratefully acknowledge. Oscar RieraLizarazu introduced me to the ITMI mapping population and actively helped in our search to identif' quantitative trait loci for resistance to cephalosporium stripe. Lynda M. Ciuffetti introduced me to laboratory techniques and allowed me to use her lab equipments throughout the study; her contributions to the project and generous help are also gratefully acknowledged. I would like to express my sincere thanks to my fellow lab member, Christina Cowger, from whom I received continuous inspiration about the project and exchanged a lot of views about cephalosporium stripe, which she worked with in her Master's project. My heartfelt appreciation also goes to Molly E. Hoffer, Senior Faculty Research Assistant, who helped me in various ways. Special thanks goes to Dan Coyle who was very friendly and willing to provide maintenance support for the growth chambers. I would like to express my gratitude to my authorities at the Bangladesh Rice Research Institute, who provided the opportunity and support for me to pursue higher study. Additional financial support for the project and myself came from USDA-CSREES- SRG-STEEP, without which I could not have finished this study. I am deeply grateful to my wife, who consistently inspired and encouraged me towards completion of the study. I also appreciate the help of newly-joined Research Assistants in Christopher C. Mundt's lab, Anne Bureau and LaRae Wallace. I would like to thank the staff and students of Lynda M. Ciuffetti and Tom Wolpert's lab for their friendship and support. CONTRIBUTION OF AUThOR Dr. Oscar Riera-Lizarazu was involved in the analysis of ITMI mapping progeny evaluation data. He also assisted in the interpretation of data. TABLE OF CONTENTS I. iNTRODUCTION .1 Importance of the disease .1 Pathogenbiology ............................................................................. 2 Influence of environment on disease development ......................................3 Role of inoculum on disease development ................................................4 Conditions affecting survival of inoculum ................................................5 InfectionProcess .............................................................................7 Resistance source and mechanism of resistance ..........................................9 Potential control measures .................................................................12 Role of toxin in cephalosporium stripe development ..................................15 Literaturecited ............................................................................... 17 II. SENSITiVITY OF WHEAT GENOTYPES TO A TOXIC FRACTION PRODUCED BY CEPHALOSPOPRIUM GRAM1NEUM AND CORRELATION WITH DISEASE SUSCEPTIBILITY ......................................................................... 25 Abstract ......................................................................................26 Introduction ...................................................................................27 Materials and Methods ......................................................................29 Laboratoryassays ............................................................................29 Extraction of toxic fraction ........................................................... 30 Assaymethod ........................................................................... 32 Statistical analyses ..................................................................... 33 TABLE OF CONTENTS (Continued) Fieldexperiments ........................................................................... 34 Locations and experimental design ..................................................34 Data collection and statistical analysis ...............................................35 Mapping progeny evaluation ..........................................................36 Results ....................................................................................... 38 Reaction of genotypes to toxic fraction .............................................. 38 Fieldreactions ........................................................................... 38 Comparison of laboratory assay to field data ........................................ 40 Toxin sensitivity
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