THESIS CHARACTERIZING THE PINE WILT DISEASE PATHOSYSTEM IN THE FRONT RANGE REGION OF COLORADO Submitted by David Atkins Department of Forest & Rangeland Stewardship In partial fulfillment of the requirements For the Degree of Master of Science Colorado State University Fort Collins, Colorado Spring 2020 Master’s Committee Advisor: Seth Davis Co-advisor: Jane Stewart Diana Wall Wade Tinkham Copyright by David H. Atkins 2020 All Rights Reserved ABSTRACT CHARACTERIZING THE PINE WILT DISEASE PATHOSYSTEM IN THE FRONT RANGE OF COLORADO Pine wilt disease, caused by the pinewood nematode (Bursaphelenchus xylophilus; PWN), is one of the most damaging invasive species in Asia. Tree mortality associated with PWN has recently been reported in Colorado and surrounding states. There remains little documentation on PWN incidence in native pine species or the biology of potential vectors in the Rocky Mountain region. Here we regionally surveyed for PWN in host trees and two putative insect vectors (Monochamus clamator & M. scutellatus) for two years to develop vector flight phenology models and test the hypothesis that disturbance factors predict vector abundance across the landscape. Flight phenology was similar between vectors: flight initiated in mid-July and continued into October for both species. PWN was distributed throughout the Front Range at rates lower than those reported in the putative native range (Host: 3.6%; Vector 4.2%). Infection rate in hosts varied among sites (0-89%), and four ‘epicenters’ of vector infectivity were identified. We also report the first incidence of PWN-M. clamator association in the U.S. The four identified epicenter sites varied in the timing of anomalous infection frequency, and flight phenology of infective vectors differed between epicenter and peripheral sites. Monochamus populations were found primarily in natural forest areas and seasonally migrate in ii small numbers to urban areas. Landscape factors such as proximity to burned area were positively correlated with Monochamus abundance. Synthesis and applications: Our study describes PWN infection frequency to be greater than that expected of a newly introduced pathogen, but lesser than the eastern United States and Canada where PWN is known to be established. Our findings provide tools that can predict exposure windows of disease exposure, which were observed to be highest in the early season in Colorado. We also describe the threat that populations of PWN in wildland forests pose to urban landscapes, and how this risk varies seasonally. These findings collectively serve characterize PWN distribution in the native ecosystem and provide tools that can be used by decision-makers and managers to proactively manage the spread of pine wilt disease. iii ACKNOWLEDGEMENTS Thank you to those who served as techs and helped out with your time to complete this project, namely Abbi, Ben, Frank, and Lillian. Thank you to my committee members Drs. Diana Wall and Wade Tinkham for your encouragement and helpful feedback as this project progressed. Your input helped shape some of the more critical aspects of this project and facilitated its completion. Thank you to those who helped me begin what I hope will be a long career in research, Drs. Brett Mattingly and Matthew Graham at ECSU and Dr. Jason Fridley at Syracuse University. Someday I will stop asking you for letters of recommendation. I would like to thank all members of the Davis (Isaac, Andrew, Khum, and Ryleigh) and Stewart (John, Brad, Sean, Jessa, Stephan, Frank, Jonathan, Jorge, Kris, Rachel, Lillian, Venezia, Emily, and Hope) labs for their company – in the field, in the lab, and as friends. I appreciate all of your time taken both to assist with the completion of this project and to provide feedback as this manuscript has developed. Thank you to my advisors Drs. Seth Davis and Jane Stewart. Your guidance has proved invaluable throughout this project, and I am a better scientist for having worked with you. Thank you for welcoming me into your labs and gently guiding me away from scientific blunders I appeared determined to make. Jane, because of the time I spent in your lab I no longer believe molecular work is mostly witchcraft. I suppose science can be done while wearing nitrile gloves after all. Thanks for your advice and for being so friendly and welcoming. Seth, thanks for showing me that even retired punks can become scientists – and good ones at that. I appreciate all of your advice and how you were willing to bring me into the fold. I will always be grateful to you for giving me this opportunity. Thank you to my friends and family. It was hard moving away to pursue this opportunity, but you made sure that I never felt far away. Thank you for your constant love and support, particularly Mom, Dad, and Mikey. Thank you to my partner Kristin. You have always been supportive of me while I spend long hours in pursuit of what is a rather abstract goal. I appreciate your encouragement and patience throughout our time together. Your companionship has made this part of the journey all the more special. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................................................. ii ACKNOWLEDGEMENTS ......................................................................................................................... iv Chapter 1 – Literature Review ................................................................................................................... Introduction ...................................................................................................................................... 4 Pine wilt disease in Asia and Europe ............................................................................................... 4 Pine wilt disease in North America ................................................................................................. 8 Pine wilt disease pathosystem ........................................................................................................ 10 Vector biology and ecology ........................................................................................................... 13 Problem statement and knowledge gaps ........................................................................................ 15 Chapter 2 – Description of pinewood nematode distribution and identification of disease epicenters ..... 22 Introduction .................................................................................................................................... 23 Materials & methods ...................................................................................................................... 26 Study design & site description ........................................................................................ 26 Data analysis ..................................................................................................................... 29 Results ............................................................................................................................................ 32 Discussion ...................................................................................................................................... 34 TABLES & FIGURES ................................................................................................................................ 46 APPENDIX ................................................................................................................................................. 55 v CHAPTER 1 LITERATURE REVIEW 1.1 Pine wilt disease: an introduction Pine wilt disease is a lethal wilting disease in susceptible conifer species caused by the pinewood nematode (Bursaphelenchus xylophilus Steiner and Nickle; hereafter, PWN). The nematode reproduces rapidly after introduction into a host tree causing disease symptoms that can appear in 2-3 weeks. Initial symptoms include needle chlorosis and wilting, decreased rate of transpiration, and reduced oleoresin flow which progress rapidly to hydraulic failure and mortality in as little as two months for extremely susceptible hosts. Pine wilt disease system consists of three key trophic levels; the PWN, insect vectors (primarily Cerambycid beetles), and the fungi upon which the nematodes feed. Blue- and black-stain Ophiostomatoid fungi are commonly associated with PWN-killed pines. These fungi are also one of the best naturally occurring substrates for PWN-growth compared to other co-occurring fungi (Kobayahsi, Sasaki, & Mamiya 1974, 1975; Sriwati et al. 2007). The fungi serve as a food source for the nematode after the resources of the host tree are exhausted post-mortality (Fukushige 1991). PWN has an obligate vector relationship with long-horned beetles (Coleoptera; Cerambycidae), specifically the pine sawyer beetles of the genus Monochamus. Monochamus spp. are cosmopolitan and sympatric with host pines throughout the northern hemisphere. These beetles are typically secondary pests in their native range, however their relationship with PWN has greatly increased interest into their biology. Here, I review the literature regarding the PWN pathosystem and disease ecology along with the resulting impacts of PWN in its introduced and native ranges and address knowledge gaps that have become apparent with emphasis on recent work that has been done here in the Front Range Region of Colorado. 1.2 Pine wilt in Asia and Europe Beginning