AN ABSTRACT OF THE THESIS OF Joseph M. Kaser for the degree of Master of Science in Entomology presented on December 1, 2009. Title: Epichloë typhina (fungus) - Botanophila lobata (fly) Interaction: An Invasive "Pollinator" System in its Introduced Range in Western Oregon. Abstract approved: _____________________________________________________________________ Sujaya Rao Epichloë typhina (Ascomycetes: Clavicipitaceae) is an endophytic fungus that infects perennial Pooid grasses and is the causal agent of choke disease. It is endemic to Europe and was inadvertently introduced into orchardgrass seed production fields in western Oregon. Choke disease, which was first recorded in Oregon in 1996, currently infects ~90% of cultivated orchardgrass seed fields in the region, resulting in yield losses >65%. Infective propagules (i.e. ascospores) are produced sexually by the bipolar heterothallic fungus and gamete outcross has been shown to be facilitated in the wild by Botanophila spp. (Diptera: Anthomyiidae), including Botanophila lobata. The fly - fungus interaction is generally considered to be one of obligatory mutualism based on studies conducted in areas endemism. However, recent evidence suggests that the fungus is able to sexually outcross in cultivated Oregon orchardgrass fields without the aid of fly "pollinators." Additionally, ascosporic fertilization has recently been implicated as an alternative mechanism for gamete transfer and might have important impacts on fungal reproduction in Oregon. The objectives of this study were to: 1) explore how two tightly linked species, which appear to have an obligate mutualistic relationship in areas of endemism, interact in a non-native context; 2) quantitatively examine the seasonal and diurnal presence of E. typhina ascospores in a cultivated Oregon orchardgrass field; and 3) test alternative transfer mechanisms of fungal spermatia for E. typhina. To address objective 1), the spatial variability and reproductive success of E. typhina and B. lobata were estimated during surveys of ten cultivated orchardgrass fields in 2008 and four fields in 2009. Fungal distributions were spatially aggregated at five of the study sites in 2008 and three in 2009. Fly distributions were spatially aggregated at three sites in 2008 and one in 2009. Botanophila lobata density exhibited a positive linear relationship with E. typhina density, suggestive of positive density dependence of fly oviposition with fungal density. However, fungal reproductive success was not affected by fly density or fungal density within the range of distributions observed in this study. To address objective 2), airborne ascospores were monitored in a single cultivated orchardgrass field during 2008 and 2009 using a Burkard volumetric spore trap. Ascospore production began in early to middle May and continued into late July during both years of the study. Daily ascospore production exhibited a circadian rhythm, with production peaking on average at 1:08 am and 12:36 am, in 2008 and 2009, respectively. The prolonged duration and high intensity of ascospore production during the growing season suggest a large window within which new plants are at risk to infection, and within which preventative management strategies must be adopted. To address objective 3), splash fertilization, contact fertilization and B. lobata fertilization were tested in the greenhouse. Although only two replicates were completed, both contact and splash fertilization appeared to be viable mechanisms of sexual outcross for E. typhina. The results of this study strongly indicate that E. typhina can successfully reproduce without the presence of B. lobata. Splash and contact fertilization, as well as ascosporic fertilization, provide opportunities for reproduction of the fungus in absence of fly pollinators. It appears that the fly - fungus interaction has shifted from an obligatory mutualism to facultative mutualism or simple fungivory within the introduced range in western Oregon. ©Copyright by Joseph M. Kaser December 1, 2009 All Rights Reserved Epichloë typhina (fungus) - Botanophila lobata (fly) Interaction: An Invasive "Pollinator" System in its Introduced Range in Western Oregon by Joseph M. Kaser A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented December 1, 2009 Commencement June 2010 Master of Science thesis of Joseph M. Kaser presented on December 1, 2009. APPROVED: _____________________________________________________________________ Major Professor, representing Entomology _____________________________________________________________________ Director of the Entomology Graduate Program _____________________________________________________________________ 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. _____________________________________________________________________ Joseph M. Kaser, Author ACKNOWLEDGEMENTS I would like to express sincere appreciation my major professor, Dr. Sujaya Rao. I would not have made it this far without her dedicated advice and insightful criticism. I thank my graduate committee members, Dr. Elizabeth Borer, Dr. Peter McEvoy and Dr. Patricia Muir, whose enthusiasm and support of this thesis was invaluable. I would like to show my gratitude to Dr. Steve Alderman from the USDA ARS National Forage Seed Production Research Center in Corvallis for sharing his time and expertise with me throughout my degree at Oregon State University. I would like to thank Kim Skyrm, Jen Bergh, Sarah Maxfield-Taylor, Missie Scherr, Melissa McKenney, Kathy Ackerman, George Hoffman, and Angela Gadino for helping with my research and making this process more than just a thesis. Thanks to Dr. Joey Spatafora and Ryan Kepler for their help with DNA extraction and PCR. Thanks to the Orchardgrass Commission, Oregon Grass Seed Council, Grass Seed Cropping Systems for Sustainable Agriculture for funding this research. And I would like to thank Chelsea Pearsall. I owe her one, big time. Finally, I would like to thank my parents, Ruth and Steve Kaser because I know they would be happy with me whether I wrote this thing or not. Thank you all. CONTRIBUTION OF AUTHORS Dr. Steve C. Alderman assisted with data collection, with interpretation of the data and with the writing of Chapter 3. Dr. Sujaya Rao assisted with interpretation of the data and with design and writing of Chapter 2, Chapter 3 and Chapter 4. LIST OF TABLES Table Page 2.1. Year of survey and age of field since original planting for sites included in the study……………………………………………………………42 2.2. Incidence of E. typhina and presence of B. lobata in 10 orchardgrass fields surveyed in 2008 (a) and 4 sites surveyed in 2009 (b)………………………………………………………………………...43 2.3. Spatial distribution of E. typhina and B. lobata in orchardgrass fields sampled in 2008 and 2009………………………………...44 2.4. Simple linear regression results of the dependent variable, flies per stroma, against stroma density in 2008 (a) and 2009 (b). Analysis is presented for two scales of observation: at the quadrat scale and the host plant scale. Sites with β1 estimates having P < 0.05 are shown in bold.…………………………………………...……….45 LIST OF FIGURES Figure Page 2.1. Abundance of B. lobata flies per infected plant in 2008 (a) and 2009 (c). Average number of flies per stroma per infected plant in 2008 (b) and 2009 (d). Frequency is equal to the total number of plants in each level on x-axis.…………………………………….……………46 2.2. νi plots depicting spatial aggregation in sites 1-4 in 2008 and 2009. Within each box, from left to right: spatial pattern of count data for infections per quadrat, stromata per quadrat and flies per quadrat are depicted. Dots represent quadrat locations. The νi values < -1.5 represent gaps (light grey); values between -1.5 and 1.5 represent random spatial arrangements (dark grey); values > 1.5 represent clusters (black). The black lines visible within each box represent field borders.…………………………………………………………47 2.3. Sites 5 through 10, surveyed in 2008, depicting νi plots of infections per quadrat, stromata per quadrat, and flies per quadrat. Dots represent actual quadrat locations. See Figure 2 (above) for an explanation of the legend. The black lines visible within each box represent field borders. …………………………………………………..48 2.4. The combined data from 2008 and 2009 for each site plotted against age of field, for: a) Proportion of plants infected in each quadrat; b) Mean stromata per host for each quadrat; and c) Mean flies per stroma for each quadrat. Open circles indicate sites from 2008, and filled circles indicate 2009. Standard error bars are depicted. …………………………………….………………………………….49 2.5. Quadrat Scale: Density of stromata per quadrat (x-axis) in relation to fly per stroma density for each quadrat (y-axis) for 2008 (a) and 2009 (b). Simple linear regression line is shown. See Table 4 2 for β1, r , and P-values.……………………..…………………………………..50 2.6. Plant Scale: Density of stromata per host (x-axis) in relation to fly per stroma density for each host (y-axis) for 2008 (a) and 2009 (b). 2 Simple linear regression line is shown. See Table 4 for β1, r , and P-values………………………………………………………………………...51 3.1. Burkard 7-day volumetric spore trap placed in an orchardgrass field used for capture of airborne ascospores.………………………………….65 LIST OF FIGURES (Continued) Figure Page 3.2. Hourly counts of E. typhina