The Pennsylvania State University The Graduate School Department of Entomology A to ZYMV GUIDE TO ERWINIA TRACHEIPHILA INFECTION: AN ECOLOGICAL AND MOLECULAR STUDY A Dissertation in Entomology by Lori Shapiro 2012 Lori Shapiro Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2012 The thesis of Lori Shapiro was reviewed and approved* by the following: Mark Mescher Assistant Professor of Entomology Thesis Advisor Chair of Committee Consuelo De Moraes Professor of Entomology Andy Stephenson Distinguished Professor of Biology and Associate Dean of Graduate Studies Shelby Fleischer Professor of Entomology Eric Harvill Professor of Microbiology and Infectious Disease Gary Felton Head of the Department of Department or Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT Many of the most ecologically and economically important diseases of plants are transmitted by insects, and the spread of these diseases ultimately depends on complex interactions among plants, pathogens, and insect vectors. Yet, empirical evidence regarding the mechanisms by which plant pathogens are transmitted by insects is sparse. In this dissertation, I take a systems biology approach to understanding ecological and molecular interactions driving the emergence of the bacterial phytopathogen Erwinia tracheiphila, a microbe with exceptional economic importance and a highly restricted host range. Induced changes in the emission of plant volatiles—airborne chemicals released by plants that serve as key foraging cues for insects but are not well characterized in response to pathogen infection. In the field and greenhouse, I found that E. tracheiphila induced a unique volatile blend from foliage, and caused a reduction in the emission of floral volatiles. The co-occurring viral pathogen Zucchini Yellow Mosaic Virus suppressed both leaf and floral volatiles. Behavioral assays found these volatile differences had effects on vector attraction and were important for beetle recruitment to E. tracheiphila infected plants and dispersal to healthy ones, and that few beetles were recruited to virus infected plants. Controlled inoculations show that healthy and ZYMV infected plants are equally susceptible to E. tracheiphila infection, and so vector behavior is largely driving lack of coinfection of the two pathogens in the same host plant that is often documented in the field. Next, I developed a qPCR method to clarify uncertainty in rates of E. tracheiphila colonization of beetle vectors exposed by feeding on wilting leaf tissue. I found that E. tracheiphila does not colonize beetles very efficiently: While at least 80% of beetles are exposed to E. tracheiphila at both short (3 hr) and long (24 hr) exposures, the longer exposure results in significantly higher long-term retention rates. Quantitative changes in bacterial titre in frass and whole beetles also show that transmission is not simply mechanical, and that E. tracheiphila exhibits complex colonization dynamics of the iv beetle’s digestive tract characterized by attachment, growth, and shedding phases. Finally, I analyze the genome of E. tracheiphila and find it has undergone extensive recombination and pseudogenization compared to the closest sequenced relative, and contains many putatively horizontally transferred genes. Phylogenetic and comparative data suggest E. tracheiphila is undergoing rapid evolution and may have recently experienced a host jump to squash. Taken together, these studies provide an ecological mechanism for transmission from infected to healthy plants; a quantitative description of how beetles are colonized and consequences for transmission; and a genome analysis hypothesizing factors influencing E. tracheiphila emergence as a squash pathogen and persistently transmitted insect symbiont. v TABLE OF CONTENTS List of Figures .......................................................................................................................... vii List of Tables ........................................................................................................................... ix Acknowledgements .................................................................................................................. xi Chapter 1 Introduction ............................................................................................................. 1 An Historical Perspective of Cucurbit Research .............................................................. 1 References ........................................................................................................................ 12 Chapter 2 Pathogen effects on vegetative and floral odors mediated vector attraction and host exposure in a complex pathosystem ......................................................................... 18 Abstract ............................................................................................................................ 18 Introduction ...................................................................................................................... 19 Materials and Methods ..................................................................................................... 22 Results .............................................................................................................................. 28 Discussion ........................................................................................................................ 35 References ........................................................................................................................ 42 Chapter 3 Direct interactions between a viral pathogen, a bacterial pathogen, and a specialist herbivore in a shared host plant ........................................................................ 45 Abstract ............................................................................................................................ 45 Introduction ...................................................................................................................... 46 Methods ............................................................................................................................ 48 Results .............................................................................................................................. 52 Discussion ........................................................................................................................ 61 References ........................................................................................................................ 64 Chapter 4 Colonization dynamics of an insect vector by a bacterial phytopathogen .............. 67 Abstract ............................................................................................................................ 67 Introduction ...................................................................................................................... 68 Materials and Methods ..................................................................................................... 69 Results .............................................................................................................................. 73 Discussion ........................................................................................................................ 81 References ........................................................................................................................ 87 Chapter 5 Comparative genomic insights into emergence and host associations of the bacterial wilt pathogen Erwinia tracheiphila ................................................................... 92 Abstract ............................................................................................................................ 92 Introduction ...................................................................................................................... 93 vi Results/Discussion ........................................................................................................... 95 Conclusions ...................................................................................................................... 130 Methods ............................................................................................................................ 132 References ........................................................................................................................ 145 Chapter 6 Conclusions ............................................................................................................. 152 References ........................................................................................................................ 157 vii LIST OF FIGURES Figure 2-1. Field foliar volatiles ............................................................................................. 29 Figure 2-2. PCA of field foliar volatiles. ................................................................................ 30 Figure 2-3. Geenhouse foliar volatiles .................................................................................... 31 Figure 2-4. Field floral volatiles. ............................................................................................ 32 Figure 2-5. Greenhouse floral volatiles ................................................................................... 32 Figure 2-6. Beetle attraction