MOLECULAR AND HISTOLOGICAL CHARACTERIZATION OF SPHAERULINA MUSIVA- POPULUS SPP. INTERACTION A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Nivi Deena Abraham In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Department: Plant Pathology January 2017 Fargo, North Dakota North Dakota State University Graduate School Title MOLECULAR AND HISTOLOGICAL CHARACTERIZATION OF SPHAERULINA MUSIVA- POPULUS SPP. INTERACTION By Nivi Deena Abraham The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Dr. Berlin Nelson Chair Dr. Jared M. LeBoldus Dr. Robert Brueggeman Dr. Wenhao Dai Approved: 2/2/2017 Dr. Jack Rasmussen Date Department Chair ABSTRACT Sphaerulina musiva, the causal agent of leaf spot and stem canker, is responsible for critical yield loss of hybrid poplar in agroforestry. This research examined quantification of S. musiva in host tissue, and infection of leaf tissue, plus gene expression between resistant and susceptible poplar genotypes. This study reports the first use of a multiplexed hydrolysis probe qPCR assay for faster and accurate quantification of S. musiva in inoculated stems of resistant, moderately resistant and susceptible genotypes of hybrid poplar at three different time points -1 wpi (weeks post-inoculation ), 3 wpi and 7 wpi. This assay detected significant differences in the level of resistance among the different clones at 3 wpi (p < 0.001) and significant differences among isolates at 1 wpi (p < 0.001), that were not detected by visual phenotyping. Histological and biochemical comparisons were made between resistant and susceptible genotypes inoculated with conidia of S. musiva in order to study the mode of leaf infection and defense response of hybrid poplar. Leaf infection was examined at 48 h, 96 h, 1 wpi, 2 wpi and 3 wpi using scanning electron microscopy (SEM) and fluorescent and laser scanning confocal microscopy. Infection process of S. musiva on Populus spp. was further characterized by transforming S. musiva with red fluorescent protein through Agrobacterium tumefaciens. Results indicated that there was no difference in pre-penetration processes, however, differences were observed in post-penetration between resistant and susceptible genotypes. The host response was also studied by examining the accumulation of hydrogen peroxide (H2O2) using fluorescent microscopy after DAB staining, and a significant difference (p < 0.0001) was observed by 2 wpi. The molecular mechanism underlying host-pathogen interaction was elucidated by studying temporal differentially expressed genes of both the interacting organisms, simultaneously, using RNA-seq. Genes involved in cell wall modification, antioxidants, antimicrobial compounds, signaling pathways, iii ROS production and necrosis were differentially expressed in the host. In the pathogen, genes involved in CWDE, nutrient limitation, antioxidants, secretory proteins and other pathogenicity genes were differentially expressed. The results from this research provide an improved understanding of poplar resistance/susceptibility to S. musiva. iv ACKNOWLEDGEMENTS I take this opportunity to express my deep sense of gratitude to my research supervisors Dr. Jared LeBoldus and Dr. Berlin Nelson for providing me unflinching encouragement and support. Their truly scientific intuition has made them a constant oasis of ideas, which exceptionally inspire and enrich my growth as a student, a researcher and the scientist I want to be. Dr. Nelson thank you once again for taking care of me as your own. I gratefully acknowledge Dr. Robert Brueggeman and Dr. Wenhao Dai for evaluation of my thesis and suggestions during my work. My special thanks to Dr. Periasamy Chithrampal, for his guidance, support and inspiration. Special thanks to Christine Ngoan and Abdullah Alhashel for helping me in formatting my thesis. And also for always being there for me. I would like to acknowledge the North Dakota State University for offering me the PhD fellowship. I am extremely grateful to my parents, sister and my husband for having patience, affection and giving me moral support. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii ACKNOWLEDGMENTS .............................................................................................................. v LIST OF TABLES ......................................................................................................................... x LIST OF FIGURES ...................................................................................................................... xi LIST OF APPENDIX TABLES ................................................................................................. xiii LIST OF APPENDIX FIGURES ................................................................................................ xiv CHAPTER 1. LITERATURE REVIEW ........................................................................................ 1 Populus Genus ........................................................................................................................... 1 Life History ................................................................................................................................ 1 General morphology .............................................................................................................. 1 Habitat ................................................................................................................................... 3 Biology .................................................................................................................................. 4 Hybrids .................................................................................................................................. 7 Populus – A Model Forest Tree ................................................................................................. 8 Diseases of Populus ................................................................................................................... 9 Sphaerulina musiva .................................................................................................................. 11 Sphaerulina musiva and the Populus Pathosystem .................................................................. 13 Management ............................................................................................................................. 17 References ................................................................................................................................ 22 CHAPTER 2. DETECTION AND QUANTIFICATION OF HYBRID POPLAR STEMS INFECTED WITH SPHAERULINA MUSIVA USING QPCR .................................................... 44 Introduction .............................................................................................................................. 44 Materials and Methods ............................................................................................................. 46 vi Primer design and validation for Sphaerulina musiva ........................................................ 46 Primer design and validation for hybrid poplar .................................................................. 47 Probe design ........................................................................................................................ 47 Multiplexed qPCR assay efficiency .................................................................................... 48 Phenotyping assay ............................................................................................................... 49 Hybrid poplar propagation ............................................................................................. 49 Pathogen propagation ..................................................................................................... 50 Experimental design, wound inoculation and DNA extraction ...................................... 51 Testing of field samples ...................................................................................................... 52 Statistical analysis ............................................................................................................... 53 Results ...................................................................................................................................... 54 Taqman qPCR assay development ...................................................................................... 54 Primer specificity and validation .................................................................................... 54 Testing qPCR assay efficiency ....................................................................................... 55 Phenotyping assay ............................................................................................................... 56 qPCR assay .......................................................................................................................... 58 Correlation of disease severity with IC values .................................................................... 59 Detection of pathogen in field samples ..............................................................................