AN ABSTRACT OF THE THESIS OF Katarina Sweeney for the degree of Master of Science in Botany and Plant Pathology presented on August 8, 2013. Title: Histological Comparisons of Needle Tissues of Four Species of White Pine infected by Cronartium ribicola. Abstract approved: ________________________________________________________________________ Jeffrey K. Stone Hypersensitive response-like (HR-like) needle reactions to infection by the white pine blister rust pathogen, Cronartium ribicola, have been reported for several species of five needle pines native to western North America. The best-studied examples are in Pinus monticola and P. lambertiana. In these species a “needle spot” phenotype has been identified in which HR-like needle reactions are related to disease resistance that is conditioned by a major gene. Conventionally it is believed that the HR-like needle reactions in resistant pines prevent spread of the pathogen to vascular stem tissue by HR mechanisms commonly seen in other plant-pathogen incompatibility interactions, i.e. a rapidly induced plant cell death and subsequent localized tissue necrosis. The dead cells present a barrier to colonization by biotrophic pathogens and cause degeneration of fungal hyphae preventing further pathogen colonization. Structural analyses of early C. ribicola needle colonization in resistant Pinus spp. have shown, however, that these symptoms and their underlying physiology are fundamentally different from the clearly defined HR described in other host-pathogen systems. Contrary to the pattern of HR responses seen in most incompatible host reactions, onset of needle lesions was first seen several weeks after initial entry of C. ribicola. We observed extensive proliferation of fungal hyphae in the host and penetration of the needle endodermis and vascular tissue by the pathogen prior to the onset of a discernable HR or cell necrosis. The amount of fungal tissue present and progress of needle colonization was similar for both resistant and susceptible Pinus spp. Therefore, typical HR does not appear to function in needles as the mechanism of disease resistance in the “needle reaction” phenotype. ©Copyright by Katarina Sweeney August 8, 2013 All Rights Reserved Histological Comparisons of Needle Tissues of Four Species of White Pine Infected with Cronartium ribicola. By Katarina Sweeney A THESIS Submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented on August 8, 2013 Commencement June 2014 Master of Science thesis of Katarina Sweeney presented on August 8, 2013. APPROVED: _________________________________________________________ Major Professor, representing Botany and Plant Pathology _________________________________________________________ Head of the Department of Botany and Plant Pathology _________________________________________________________ Dean of the Graduate School 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. _________________________________________________________ Katarina Sweeney, Author ACKNOWLEDGEMENTS I would like to thank Jeff Stone and the department of botany and plant pathology for giving me the opportunity to study at Oregon State University, Ken Johnson and Tom Wolpert for valuable discussions about my research and Barb Taylor for support and practical guidance in the planning stages of my thesis. Especially, I would like to thank Kathy Cook who trained me in microtomy and histo-chemical techniques and became a true mentor during my grad school career. She always provided insightful advice whenever it was of need during the last three years. Moreover, I would like to thank Richard Sniezko, Angelia Kegley and the Dorena staff in Cottage Grove, OR for their help with my project. Finally, I would like to thank my friends for their ongoing support and everyone else who open-mindedly showed encouragement of my decision to change careers. TABLE OF CONTENTS Page 1. Introduction 1.1 Biology of the white pine blister rust pathosystem……………………………. 1 1.2 White pine blister rust symptoms and damage…………………………………5 1.3 The white pines of North America……………………………………………..8 1.4 Introduction of white pines into Europe………………………………………10 1.5 Cronartium ribicola and its invasion of North America………………………11 1.6 Review of resistance research…………………………………………………12 1.7 Three types of white pine resistance…………………………………………..15 1.8 Major gene resistance………………………………………………………….17 1.9 The hypersensitivity response…………………………………………………18 1.10 Major gene resistance in five-needle pines……………………………………19 1.11 The hypersensitivity response in five-needle pines……………………………20 1.12 Virulence genes in the blister rust pathogen…………………………………...21 1.13 Research objectives……………………………………………………………23 2. Methods 2.1 White pine seedlings and inoculations…………………………………………24 2.2 Pine needle sample processing…………………………………………………27 TABLE OF CONTENTS (continued) Page 3. Results 3.1 Structural analysis of host tissues………………………………………………29 3.2 Structural observations in Pinus flexilis………………………………………..34 3.3 Structural observations in Pinus monticola…………………………………….40 3.4 Structural observations in Pinus lambertiana………………………………….45 3.5 Structural observations in Pinus albicaulis…………………………………….49 4. Discussion 4.1 Histology and structural analyses………………………………………………54 4.1.1 Pinus flexilis……………………………………………………………...54 4.1.2 Pinus monticola…………………………………………………………..55 4.1.3 Pinus lambertiana………………………………………………………..56 4.1.4 Pinus albicaulis…………………………………………………………..57 4.2 Integration of histological findings…………………………………………….58 5. Conclusions………………………………………………………………………….69 Bibliography……………………………………………………………………………70 LIST OF FIGURES Page Figure 1. Aecial stage of white pine blister rust on western white pine………………………..2 2. Pycnial Stage of C. ribicola on P. monticola...............................................................3 3. The life cycle of white pine blister rust………………………………………………5 4. Foliage of susceptible P. lambertiana 97 days after initial inoculation……………...6 5. Foliage of susceptible P. albicaulis 97 days after initial inoculation………………...6 6. Early bark discoloration symptom of C. ribicola stem infection on P. monticola…...7 7. The underside of a Ribes nigrum leaf infected with C. ribicola……………………..26 8. The inoculation chamber at Dorena Genetic Resource Center 1…………………….26 9. The inoculation chamber at Dorena Genetic Resource Center 2…………………….26 10. The inoculation chamber at Dorena Genetic Resource Center 3…………………...26 11. Cross section of P. albicaulis needle with C. ribicola haustorium………………....29 12. Drawing of C. ribicola haustorium by Reginald H. Colley, 1917………………….29 13. Cross section of an uninoculated P. monticola needle, visual score “0”…………...31 14. Cross section of P. flexilis needle infected with C. ribicola, Visual score “1”…….31 15. Cross section of P. albicaulis needle infected with C. ribicola, Visual score “1,2,4”…………………………………………….…32 LIST OF FIGURES (continued) Page 16. Cross section of P. albicaulis needle infected with C. ribicola, Visual score :“1,2,3,4,5”…………………………………………..32 17. Cross section of P. lambertiana needle infected with C. ribicola, Visual score :“1,4,5”………………………………………………33 18. Cross section of P. flexilis needle infected with C. ribicola, Visual score :“5”…………………………………………………..33 19. Picture plate, P. flexilis, resistant……………………………………………….......35 20. Picture plate, P. flexilis, and susceptible ……………………………………….......35 21a. Histology P. flexilis, resistant………………………………………………...........39 21b. Histology P. flexilis, susceptible…………………………………………………..39 22. Picture plate, P. monticola, resistant……………………………………………......41 23. Picture plate, P. monticola, susceptible…………..…………………………….......41 24a. Histology P. monticola, resistant………………………………………………….44 24b. Histology P. monticola, susceptible………………………………………………44 25. Picture plate, P. lambertiana, resistant…………………………………………….46 26. Picture plate, P. lambertiana, susceptible………………………………………….46 27. Histology P. lambertiana…………………………………………………………..48 28. Picture plate, P. albicaulis resistant…………………..…………………………....50 29. Picture plate, P. albicaulis susceptible……………………………………………..50 30a. Histology P. albicaulis resistant…………………………………………………..53 30b. Histology P. albicaulis susceptible……………………………………………….53 LIST OF TABLES Page Table 1. Spore stages of Cronatium ribicola and their characteristics………………………..1 2. Taxonomy and nomenclature of the genus Pinus Lemm. ………………………….8 3. Gene-for-gene interaction for plant host and pathogen after Flor (1942)…………...22 4. Seedlings used for needle collections………………………………………………..25 5. P. flexilis needle samples…………………………………………………………....36 6. P. monticola needle samples………………………………………………………..42 7. P. lambertiana needle samples……………………………………………………..47 8. P. albicaulis needle samples………………………………………………………..51 Dedicated to Kathy Cook 1 1. INTRODUCTION 1.1 Biology of the White Pine Blister Rust Pathosystem White pine blister rust (WPBR) is caused by the Pucciniomycete pathogen Cronartium ribicola (Cronartiaceae) J.C. Fischer (Rabenhorst, 1872). The obligate biotroph exhibits five spore stages (TABLE 1) and requires two unrelated species as host plants to complete its life cycle. Only five-needle pines (Pinaceae, subsection Strobus) are susceptible to infection by C. ribicola and serve as the spermatial (via pycniospores) and the aecial host for the pathogen; Ribes spp. (Grossulariaceae) are the main uredinial