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Sudden Oak Death Science Symposium III

Santa Rosa, California March 5 ­ 9, 2007

DISCLAIMER Papers were provided by the authors in camera-ready form for printing. Authors are responsible for the content and accuracy. Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture. The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service.

PESTICIDE PRECAUTIONARY STATEMENT This publication reports research involving pesticides. It does not contain recommendations for their use, nor does it imply that the uses discussed here have been registered. All uses of pesticides must be registered by appropriate state or federal agencies, or both, before they can be recommended. CAUTION: Pesticides can be injurious to humans, domestic animals, desirable plants, and fish or other wildlife if they are not applied properly. Use all pesticides selectively and carefully. Follow recommended practices for the disposal of surplus pesticides and pesticide containers.

TABLE OF CONTENTS

Welcome..............................................................................................................................1 Conference Sponsors .........................................................................................................2 Additional Program Committee Members......................................................................2 Program of Events Overview............................................................................................3 ORAL PRESENTATIONS Susceptibility to Phytophthora ramorum in California Bay Laurel, a Key Foliar Host of Sudden Oak Death Brian Anacker .....................................................................................................................5 Pathogenicity of Phytophthora Species Isolated from Soil in the Eastern United States Yilmaz Balci ........................................................................................................................6 Infection of Tree Stems by Zoospores of Phytophthora ramorum and P. kernoviae Clive Brasier .......................................................................................................................8 Phytophthora ramorum + P. kernoviae = International Biosecurity Failure Clive Brasier .....................................................................................................................10 Population Structure of Phytophthora ramorum in Oregon Jennifer Britt .....................................................................................................................11 Invasion of Xylem of Mature Tree Stems by Phytophthora ramorum and P. kernoviae Anna Brown ......................................................................................................................12 Recovery of Phytophthora ramorum from Mistletoe and California Bay Inflorescences and Possible Implications Relating to Disease Spread Gary Chastagner ..............................................................................................................13 Spread and Development of Phytophthora ramorum in a California Christmas Tree Farm Gary Chastagner ...............................................................................................................14 Detection and Quantification of mRNA by Reverse Transcription Real-Time PCR as an Indicator of Viability in Phytophthora ramorum Infected Soil and Plant Material Antonio Chimento .............................................................................................................16

Stand Level Infection and Mortality Dynamics in Phytophthora ramorum Infested Redwood-Tanoak Forests: Patterns and Predictions Based on Five Years of Monitoring Richard C. Cobb ...............................................................................................................17 Landscape Connectivity Influences the Establishment of Phytophthora ramorum T. Emiko Condeso .............................................................................................................18 Human Activity and the Spread of Phytophthora ramorum J. Hall Cushman ................................................................................................................19 Effect of Environmental and Seasonal Factors on the Susceptibility of Different Rhododendron Species and Hybrids to Phytophthora ramorum Isabelle De Dobbelaere ....................................................................................................21 Sporulation of Phytophthora ramorum and P. kernoviae on Asymptomatic Foliage Sandra Denman ................................................................................................................23 Genotyping Indicates Nursery and Early Phytophthora ramorum Collections are Indistinguishable, while Showing Current Local Diversification of Wild Populations Driven by Geography Matteo Garbelotto .............................................................................................................24 Vegetation Response Following Phytophthora ramorum Eradication Treatments in Southwest Oregon Forests Ellen Goheen .....................................................................................................................25 What can Availability of the Phytophthora ramorum Genome do for us? Niklaus Grünwald .............................................................................................................27 Linking Sudden Oak Death Risk Models with Economic Impact Assessments Thomas Holmes .................................................................................................................28 Predicting the Spread of Sudden Oak Death in California: Spatio-Temporal Modeling of Susceptible-Infectious Transitions Richard Hunter .................................................................................................................30 Detecting Phytophthora ramorum and Other Species of Phytophthora in Streams in Natural Ecosystems Using Baiting and Filtration Methods Jaesoon Hwang .................................................................................................................32 Eradication of Phytophthora ramorum from Oregon Forests: Status after Six Years. Alan Kanaskie ...................................................................................................................34

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Introducing the Phytophthora Database: An Integrated Resource for Detecting, Monitoring, and Managing Phytophthora Diseases Seogchan Kang .................................................................................................................35 Assessment of Potential Economic and Environmental Impacts Caused by Phytophthora ramorum in Europe Hella Kehlenbeck ..............................................................................................................37 Revision of the USDA Forest Service National Sudden Oak Death Risk Map: Improved Procedures for Data Mining, Risk Mapping, and Modeling Frank Koch .......................................................................................................................38 Climate-Host Mapping of Phytophthora ramorum, Causal Agent of Sudden Oak Death Roger Magarey .................................................................................................................40 Mitochondrial Genomics in the Genus Phytophthora with a Focus on Phytophthora ramorum Frank Martin .....................................................................................................................41 Predicting Movement of Nursery Hosts Using a Linear Network Model Steve McKelvey .................................................................................................................43 Attraction of Ambrosia and Bark Beetles to Coast Live Oaks Infected by Phytophthora ramorum Brice McPherson ..............................................................................................................45 Influence of Historical Woodland Expansion on the Establishment of Phytopthora ramorum Ross Meentemeyer ............................................................................................................47 Log Susceptibility of Iberian Tree Species to Phytophthora ramorum Eduardo Moralejo .............................................................................................................49 Distribution of Phytophthora ramorum, P. nemorosa, and P. pseudosyringae in Native Coastal California Forest Communities Shannon Murphy ...............................................................................................................51 2006 Pilot Survey for Phytophthora ramorum in Forest Streams in the USA Steven Oak ........................................................................................................................53 Chemistry of Coast Live Oak Response to Phytophthora ramorum Infection Frances Ockels ..................................................................................................................55 The Ecology of Phytophthora ramorum in Ireland Carmel O' Connor .............................................................................................................56 iii

Phytophthora ramorum Infects Sapwood and is Associated with Reduced Specific Conductivity of Xylem Vessels in Tanoak Jennifer Parke ...................................................................................................................57 Epidemiological Modeling of Phytophthora ramorum: Network Properties of Susceptible Plant Genera Movements in the UK Nursery Sector Marco Pautasso ................................................................................................................59 Influence of Oak Woodland Composition and Structure on Infection by Phytophthora ramorum Nathan Rank ......................................................................................................................60 Phytophthora Species Associated with Stem Cankers on Tanoak in Southwestern Oregon Paul Reeser .......................................................................................................................62 Comparison of Phosphonate and Azomite Treatments for Control of SOD in Coast Live Oak (Quercus agrifolia). Doug Schmidt ....................................................................................................................63 Quantification of Sudden Oak Death Tree Mortality in the Big Sur Ecoregion of California Douglas Shoemaker ..........................................................................................................64 Contingency Planning for Phytophthora ramorum Outbreaks: Progress Report Work Package 7, EU RAPRA Project Maarten Steeghs ................................................................................................................66 Distance from California Bay Reduces the Risk and Severity of Phytophthora ramorum Canker in Individual Coast Live Oaks Ted Swiecki .......................................................................................................................67 Seasonal Symptom Expression, Laboratory Detection Success, and Sporulation Potential of Phytophthora ramorum on Rhododendron and Camellia Steve Tjosvold ...................................................................................................................69 Natural Outbreaks of Phytophthora ramorum in the UK - Current Status and Monitoring Update Judith Turner ....................................................................................................................71 The OakMapper WebGIS: Improved Access to SOD Data Karin Tuxen ......................................................................................................................72 Four Years of Experience with Filtration Systems in Commercial Nurseries for Eliminating Phytophthora Species from Recirculation Water Thorsten Ufer ....................................................................................................................74 iv

Refining the Detection of Phytophthora ramorum: What PCR Kinetics can Tell Pedro Uribe ......................................................................................................................75 Wildland Management of Phytophthora ramorum in Northern California Forests Yana Valachovic ...............................................................................................................76 Monitoring for Phytophthora ramorum and Other Species of Phytophthora in Nurseries and Urban Areas in the Southeastern USA Yeshi Wamishe ..................................................................................................................77 Dissemination of Aerial and Soilborne Phytophthoras by Human Vectors Joan Webber .....................................................................................................................79 Status of Phytopthora ramorum and P. kernoviae in Europe Joan Webber .....................................................................................................................80 Can Phytophthora ramorum be Spread with Contaminated Irrigation Water? Sabine Werres ...................................................................................................................81 Soil Treatments for the Elimination of Phytophthora ramorum from Nursery Beds: Current Knowledge from the Laboratory and the Field. Lani Yakabe ......................................................................................................................82

POSTERS Phytophthora ramorum Inoculum Potential on California Bay Laurel (Umbellularia californica): Individual Leaves Support Repeated Inoculum Production Shelley Arnold ...................................................................................................................84 Effectiveness of Fungicides in Protecting Conifers and Rhododendrons from Foliar Infection by Phytophthora ramorum Gary Chastagner ...............................................................................................................86 Microbial- and Isothiocyanate-Mediated Control of Phytophthora and Pythium Species Michael Cohen ..................................................................................................................88 Spatial and Temporal Aspects of Tylosis Formation in Tanoak Inoculated with Phytophthora ramorum Brad Collins ......................................................................................................................90 Estimating the Economic Impacts of Phytophthora ramorum on Washington State Nurseries Norman Dart .....................................................................................................................91 v

Effects of Environmental Variables on the Survival of Phytophthora ramorum in Bay Laurel Leaves Matthew DiLeo ..................................................................................................................93 Endophytic Fungi of California Bay Laurel Greg Douhan ....................................................................................................................95 Identification of Control Agents and Factors Affecting Pathogenicity of Phytophthora ramorum Marianne Elliott ................................................................................................................96 In vitro Testing of Biological Control Agents on A1 and A2 Isolates of Phytophthora ramorum Marianne Elliott ................................................................................................................98 New Relationships among the Sudden Oak Death Pathogen, Native Bark and Ambrosia Beetles, and Decay Fungi Colonizing Oaks Nadir Erbilgin .................................................................................................................100 Summer Survival of Phytophthora ramorum in California Bay Laurel Leaves Elizabeth Fichtner ...........................................................................................................102 Suppression of Phytophthora ramorum in Aluminum-Amended Peatmoss Elizabeth Fichtner ...........................................................................................................104 Aerial and Targeted Ground Surveys for SOD in California, 2001 through 2006 Lisa Fischer ....................................................................................................................106 Molecular Evolution of an Avirulence Homolog (Avh) Gene Subfamily in Phytophthora ramorum Erica Goss .......................................................................................................................107 Effect of Flooding on Root and Foliar Disease Severity on Rhododendron Caused by Phytophthora ramorum Niklaus Grünwald ...........................................................................................................108 Screening Different Phytophthra ramorum Hosts for Limitations of Detection Level Using Various Molecular Genetic Techniques Tamar Harnik ..................................................................................................................109 Mapping Oak Mortality for Early Detection of Phytophthora ramorum: A Comparison of Aerial Surveys and Digital Aerial Photo Interpretation Erik Haunreiter ...............................................................................................................110 Correlating Phytophthora ramorum Infection Rate and Lesion Expansion in Tanoak Katherine Hayden ...........................................................................................................111 vi

Distribution of Phytophthora ramorum in Norway Maria Luz Herrero ..........................................................................................................112 Monitoring for Phytophthora ramorum in Nevada; Our Driest State Dawn Holzer ...................................................................................................................113 Susceptibility of Selected Ornamental Plants towards Phytophthora ramorum Katrin Kaminski ..............................................................................................................114 Understanding the Spatial Component of Sudden Oak Death Maggi Kelly .....................................................................................................................115 Evaluation of a Rapid Diagnostic Field Test Kit for Identification of Phytophthora ramorum, P. kernoviae, and other Phytophthora Species at the Point of Inspection Charles Lane ...................................................................................................................116 Comparative Susceptibility of Plants Native to the Appalachian Range of the United States to Inoculation with Phytophthora ramorum Robert Linderman ...........................................................................................................117 Pathogenicity Variation in Two West Coast Forest Phytophthoras, Phytophthora nemorosa and P. pseudosyringae, to Bay Laurel Rachel Linzer ..................................................................................................................118 Monitoring Phytophthora ramorum and P. kernoviae in Soil and Rainwater Samples Collected at Two Sites in Caerhays Castle Gardens, Cornwall, UK David Lockley .................................................................................................................119 Monitoring Phytophthora ramorum in Soil, Leaf Litter, and Rain Traps at the Lost Gardens of Heligan, Cornwall, UK David Lockley .................................................................................................................120 Development of Phytophthora ramorum Infection and Disease Symptoms on Coast Redwood Seedlings Sunny Lucas ....................................................................................................................123 Antifungal Activity of Extracts and Select Compounds in the Heartwood of Seven Western Conifers toward Phytophthora ramorum Daniel Manter .................................................................................................................124 Photosynthetic Declines are Induced by Phytophthora ramorum Infection and Exposure to Elicitins Daniel Manter .................................................................................................................125

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Evaluation of Molecular Markers for Phytophthora ramorum Detection and Identification using a Standardized Library of Isolates Frank N. Martin ..............................................................................................................126 Evaluation of Infection Potential and Sporulation of Phytophthora ramorum on Six Rhododendron Cultivars. Virginia McDonald .........................................................................................................127 In vitro Foliage Susceptibility of Canary Islands Laurel Forests: A Model for Understanding the Ecology of Phytophthora ramorum Eduardo Moralejo ...........................................................................................................128 Monitoring Phytophthora ramorum Distribution in Streams within Coastal California Watersheds Shannon Murphy .............................................................................................................130 Phytophthora ramorum Early Detection Surveys for Forests in the USA 2003-2006 Steven Oak ......................................................................................................................132 Implementation of a Thinning and Burning Study in Tanoak-Redwood Stands in Santa Cruz and Mendocino Counties Kevin O'Hara ..................................................................................................................134 Contemporary California Indian Uses of the Regulated Hosts and Associated Species Affected by Phytophthora ramorum Beverly Ortiz ...................................................................................................................135 Global Gene Expression Profiles of Phytophthora ramorum Strain Pr102 in Response to Plant Host and Tissue Differentiation Caroline Press ................................................................................................................136 Phytophthora siskiyouensis, a New Species from Soil and Water in Southwest Oregon Paul Reeser .....................................................................................................................137 Susceptibility of Some Native Plant Species from Hawaii to Phytophthora ramorum Paul Reeser .....................................................................................................................138 Studies on the Tissue Colonisation in Rhododendron by Phytophthora ramorum Marko Riedel ..................................................................................................................139 In planta and In vitro Comparisons of Wild and Nursery Isolates of Phytophthora ramorum Noah Rosenzweig ............................................................................................................140

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Phytophthora ramorum in Scotland: It's all over? Alexandra Schlenzig ........................................................................................................141 Results of Monitoring Phytophthora ramorum in Germany since the Year 2003 Thomas Schroeder ..........................................................................................................142 Temperature-Dependent Phenotypic Variation among Phytophthora ramorum Isolates from Eastern Sonoma County Valerie Sherron ...............................................................................................................144 Environmental Parameters Affecting Inoculum Production from Lilac Leaf Pieces Infected with Phytophthora ramorum Nina Shishkoff .................................................................................................................145 Development and Germination of Phytophthora ramorum Chlamydospores Aaron Smith ....................................................................................................................146 Sudden Oak Death Risk along an Elevational Transect in the Southeastern US Pauline Spaine ................................................................................................................147 Comparing Phytophthora ramorum Diagnostic Protocols for the National SOD Stream Monitoring Program Wendy Sutton ..................................................................................................................148 Stream Monitoring for Detection of Phytophthora ramorum in Oregon Wendy Sutton ..................................................................................................................149 Preservation of Lithocarpus densiflorus Diversity on California's Central Coast a Cooperative Project with Area Residents Steven Swain ...................................................................................................................150 Survival of Phytophthora ramorum Chlamydospores at High and Low Temperatures Paul Tooley .....................................................................................................................151 A High Throughput System for the Detection of Phytophthora ramorum in Susceptible Plant Species Aaron Trippe ...................................................................................................................152 The Efficacy of Heat Treatment to Control Phytophthora ramorum in Infected Wood Species Kayimbi Tubajika ............................................................................................................154 A Comparative Analysis of Diagnostic Approaches for Phytophthora ramorum AnnaMaria Vettraino ......................................................................................................155

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Evaluation of Fungicides for Control of Phytophthora ramorum Stefan Wagner .................................................................................................................156 The Big Sur Ecoregion Sudden Oak Death Adaptive Management Project: Ecological Monitoring Allison Wickland .............................................................................................................158 Investigating the Potential of Biological Control against Phytophthora ramorum Timothy Widmer ..............................................................................................................160 Surveying for Phytophthora Species, including Phytophthora ramorum, in Soils and Water Sources from Ornamental Nurseries in Georgia Jean Williams-Woodward ...............................................................................................161

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Welcome

Welcome to the Sudden Oak Death Science Symposium III, and thank you for your participation. By bringing together scientists, regulators, industry representatives, land managers, and policy makers from around the globe, this conference is intended to renew the momentum set in motion by the first two Sudden Oak Death Science Symposiums. The research findings and ongoing projects presented this week will provide an overview of worldwide scientific information on Phytophthora ramorum, and will hopefully encourage and refresh communication, coordination, and cooperation among the various affected disciplines worldwide. With the Sudden Oak Death pathogen killing and infecting trees by the millions as well as impacting the horticultural industry globally, advances in research and management options are critical, both to the industries that trade susceptible plants and to those that work to protect the world's forests and wildlands. Susan Frankel, Sudden Oak Death Research Program Manager Pacific Southwest Research Station Albany, California [email protected] Katie Palmieri, Public Information Officer California Oak Mortality Task Force/UC Berkeley Berkeley, California [email protected] Janice Alexander, Sudden Oak Death Outreach Coordinator California Oak Mortality Task Force/UC Cooperative Extension Marin Novato, California [email protected] Mark Stanley, Chairperson California Oak Mortality Task Force/CA Department of Forestry and Fire Protection Sacramento, California [email protected]

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Conference Sponsors

USDA Forest Service Pacific Southwest Research Station Albany, California USDA Forest Service Pacific Southwest Region Vallejo, California California Oak Mortality Task Force University of California, Integrated Hardwood Range Management Program, Center for Forestry, Division of Agriculture and Natural Resources Berkeley, California

Additional Program Committee Members

Dr. Kerry Britton, USDA Forest Service Research and Development Arlington, Virginia Dr. Russ Bulluck, USDA APHIS Center for Plant Health Science Technology Raleigh, North Carolina Jonathan Jones, USDA APHIS Plant Protection and Quarantine Riverdale, Maryland

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Program of Events Overview

SUNDAY, MARCH 4, 2007 7:00 p.m. ­ 8:00 p.m. Registration

MONDAY, MARCH 5, 2007 7:30 a.m. ­ 8:30 a.m. 8:30 a.m. ­ 11:30 a.m. 11:30 a.m. ­ 12:30 p.m. 11:30 a.m. ­ 1:00 p.m. 1:00 p.m. ­ 6:00 p.m. 7:00 p.m. ­ 8:00 p.m. Registration Risks to Conifers Panel Organized by: North American Plant Protection Organization Registration Lunch (on own) Sudden Oak Death Field Trip Dinner (on own) Registration

TUESDAY, MARCH 6, 2007 7:00 a.m. ­ 8:15 a.m. 8:15 a.m. ­ 8:30 a.m. 8:30 a.m. ­ 9:00 a.m. 9:00 a.m. ­ 10:00 a.m. 10:00 a.m. ­ 10:30 a.m. 10:30 a.m. ­ 11:10 a.m. 11:10 a.m. ­ 12:15 p.m. 12:15 p.m. ­ 1:15 p.m. 1:15 p.m. ­ 2:30 p.m. 2:30 p.m. ­ 3:00 p.m. 3:00 p.m. ­ 3:45 p.m. 3:45 p.m. ­ 5:00 p.m. Registration Welcome Keynote Speaker Wildland Update Break Nursery Update Regulatory Update Lunch (provided) Landscape Monitoring and Mapping Break Landscape Monitoring and Mapping, Continued Diagnostics

5:15 p.m. ­ 7:15 p.m. California Oak Mortality Task Force Nursery Committee Meeting WEDNESDAY, MARCH 7, 2007 8:00 a.m. ­ 10:00 a.m. 10:00 a.m. ­ 10:30 a.m. 10:30 a.m. ­ 12:00 p.m. 12:00 p.m. ­ 1:00 p.m. Nursery Research and Management Break Forest Insects and Pathogens: Quarantine Issues Lunch (provided)

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1:00 p.m. ­ 2:40 p.m. ­ 3:00 p.m. ­ 5:30 p.m. ­

2:40 p.m. 3:00 p.m. 5:00 p.m. 7:30 p.m.

Biology and Ecology Break Biology and Ecology, Continued Poster Session (Social)

THURSDAY, MARCH 8, 2007 8:00 a.m. ­ 10:15 a.m. 10:15 a.m. ­ 10:45 a.m. 10:45 a.m. ­ 12:00 p.m. 12:00 p.m. ­ 1:00 p.m. 1:00 p.m. ­ 2:15 p.m. 2:15 p.m. ­ 2:45 p.m. 2:45 p.m. ­ 3:25 p.m. 3:25 p.m. ­ 4:45 p.m. 4:45 p.m. ­ 5:00 p.m. Biology and Ecology, Continued Break Genetics Lunch (provided) Modeling Break Modeling, Continued Can P. ramorum be Managed? (Landscape Management) Closing Remarks

FRIDAY, MARCH 9, 2007 Regulatory and Management Research Needs Assessment Organized by Department for Environment, Food and Rural Affairs; United States Department of Agriculture, Animal and Plant Health Inspection Service; United States Department of Agriculture, Forest Service, Pacific Southwest Research Station General Session: Objectives, process, and desired outcome for Research Needs Assessment 9:00 a.m. ­ 10:30 a.m. Concurrent Sessions Forestry Needs Assessment Horticultural Needs Assessment 10:30 a.m. ­ 11:00 a.m. Break 11:00 a.m. ­ 12:00 p.m. General Session: Report out and discussion 12:00 p.m. Adjourn 8:30 a.m. ­ 9:00 a.m.

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ORAL PRESENTATIONS

Susceptibility to Phytophthora ramorum in California Bay Laurel, a Key Foliar Host of Sudden Oak Death

Brian Anacker, UC Davis, Graduate Group in Ecology, Department of Environmental Science and Policy, Davis, CA, USA, 94616; [email protected]; Nathan Rank, Sarah Gordon, and Rich Whitkus, Sonoma State University; Daniel Huberli, Matteo Garbelotto, and Tamar Harnik, UC Berkeley Sudden Oak Death, caused by the water mold Phytophthora ramorum, is a plant disease responsible for the death of 100s of thousands of oak and tanoak trees. California bay laurel (Umbellularia californica) plays a key role in P. ramorum inoculum build-up and spread to oaks. While bay laurel appears to vary in susceptibility to P. ramorum, little is known about the causes or extent of this variability. In this research, we examine phenotypic and genotypic correlates of bay laurel susceptibility across two isolates of P. ramorum. We conducted lab susceptibility trials on detached leaf samples and assessed field symptom levels for 102 trees from 13 locations in northern California. We found variability in lesion size produced on detached leaves was significantly related to AFLP markers, suggesting a genetic basis to resistance. Susceptibility varied significantly among bay laurel trees, with a five fold difference in lesion size. The phenotypic trait of leaf area was significantly related to lesion size, where bigger leaves produced bigger lesions. The two different isolates produced lesions that were significantly correlated across leaves, but differences in isolate virulence increased with bay laurel susceptibility. Variation in field symptom levels was explained primarily by environmental conditions. This work demonstrates that susceptibility to P. ramorum in bay laurel depends on genetic, phenotypic, and environmental characteristics, as well as P. ramorum isolate virulence, and provides useful information for predicting spread risk among bay laurel and onto oak trees.

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Pathogenicity of Phytophthora Species Isolated from Soil in the Eastern United States

Yilmaz Balci, Selin Balci, and William MacDonald, West Virginia University, Plant & Soil Sciences, 1090 South Ag. Science Build., Morgantown, WV, USA, 26506; [email protected]; Kurt Gottschalk, USDA, Northern Research Station The susceptibility of stem and foliar tissues of native oak species (Quercus spp.) to infection by seven different Phytophthora species was assessed. Species tested included P. cambivora, P. cinnamomi, P. citricola, P. europaea, P. quercina "like", P. sp1 and P. sp2. Pathogenicity tests performed included stem inoculation of 2-year-old oaks grown under field conditions and 1-year-old greenhouse seedlings. Stem inoculations were conducted in June and September and evaluated two months later. Oak species included in the stem inoculation trials included Q. bicolor, Q. macrocarpa, Q. montana, Q. palustris, Q. rubra, and Q. velutina. Foliage inoculations also were conducted on oak and understory plant species to evaluate the ability of Phytophthora species to infect foliar tissue. In addition to the six oak species tested as part of the stem inoculation experiments, foliage of Q. alba, Q. imbricara, Q. robur, Castanea dentata, Kalmia latifolia, and Rhododendron maximum were used. P. citricola and P. cinnamomi generally were able to cause significantly larger lesions than the other Phytophthora species on the 1-and 2-year-old seedlings. P. cambivora and P. europaea also produced lesions that were larger than the controls, but infection did not occur as consistently as with P. citricola and P. cinnamomi. Of all the oak species tested, Q. rubra was the least susceptible oak. However, P. cinnamomi and P. citricola were still able to produce lesions on 1-year-old Q. rubra seedlings. In general, lesion sizes were larger on 2-yearold seedlings compared to 1-year-old seedlings. For the 1-year-old seedlings there were no significant differences in lesion sizes between the June and September inoculation periods, although significant seasonal differences occurred with the 2-year-old fieldgrown seedlings. With exception of Kalmia latifolia, younger foliage of the various plant species was more susceptible to Phytophthora infection than the older. Of all the species tested, Q. rubra and Castanea dentata had the greatest foliar area invaded. Wounding had no effect on the infection of foliage of these two species but did with all the others. Other species with susceptible foliage were Q. alba and Q. robur. The least susceptible one 6

were Q. bicolor, Q. imbricara, Q. montana and Q. palustris. When Phytophthora species were ranked by their ability to cause significantly larger lesions than the controls, P. citricola was the most effective species followed in descending order by P. cinnamomi, P. europaea, P. cambivora, P. sp2 and P. quercina "like". P. citricola, and P. cinnamomi were most consistently able to invade stem and foliar tissue. The ability of other species to colonize host tissue was variable and depended on the age and type of the host, tissue type, and environmental condition. Of the various oaks tested Q. rubra had the least susceptible stem but the most susceptible foliage to Phytophthora infection.

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Infection of Tree Stems by Zoospores of Phytophthora ramorum and P. kernoviae

Clive Brasier, Anna Brown, Joan Rose, and Susan Kirk, Forest Research Agency, UK, Forest Research, Alice Holt Lodge, Farnham, Surrey, UK, GU10 4LH; [email protected] Phytophthora ramorum, P. kernoviae, and other aerial Phytopthoras cause bleeding lesions on the trunks of mature trees. Many pathogenicity tests carried out on tree stems use wound inoculation methods. However, little is known about the ability of zoospore inoculum to directly penetrate intact tree bark, although this is the presumed mode of entry of Phytophthoras above ground level. We report here research in progress on zoospore infection of tree stems using both laboratory generated and natural zoospore inoculum. In a preliminary laboratory test, P. ramorum zoospore suspensions were placed in tubes attached to unwounded surfaces of freshly cut 50 cm long x 12 cm diameter stems of several tree species using modelling clay. After two weeks the inoculum source and outer bark were removed. Lesions of several centimetres were observed in the phloem of Fagus sylvatica, Castanea sativa, Quercus rubra, Pseudotsuga menziesii, and Picea sitchensis. Lesions were absent in Q. robur but the pathogen could be re-isolated from visually healthy phloem beneath the inoculum points i.e. the bark was penetrated but no lesions developed within the time frame. In a similar experiment using logs of F. sylvatica only, P. kernoviae, P. citricola and P. cambivora all caused phloem lesions. These experiments therefore demonstrated that zoospore inoculum of four Phytophthora species resulted in penetration of unwounded bark. In field experiments, freshly cut sealed logs 60 cm long x 15 cm diameter of F. sylvatica, Q. robur, and Acer pseudoplatanus were placed under rhododendrons infected with either P. ramorum or P. kernoviae at two woodland sites. Potential inoculum was assumed to be of zoospore origin. Twelve logs of each species were used at each site, half being examined for lesion development after 6 weeks and half after 11 weeks. The experiment was carried out twice. In the first experiment established in early July 2006, hot dry conditions prevailed from July to mid August and no lesions were found. In the second 8

established in early August, rain occurred from late August onwards. Lesions caused by both Phytophthora species were found on the upper surfaces of the F. sylvatica logs only after 6 weeks. By 11 weeks the P. kernoviae exposed F. sylvatica logs averaged 3 lesions per log of mean lesion area ca. 25 cm²; and the P. ramorum logs 6.5 lesions per log of mean lesion area ca. 8 cm². The Phytophthora species were confirmed by isolation. Lesions were therefore more numerous with P. ramorum but larger with P. kernoviae. For both Phytopthoras, two-thirds of the lesions were associated with either cut ends of the logs, healed branch stubs or active epicormic shoots. The remainder showed no association. External bleeds were also associated with a number of the lesions on the F. sylvatica logs. Four lesions (but no bleeds) occurred on the six Q. robur logs exposed to P. ramorum. No lesions occurred on the Q. robur logs exposed to P. kernoviae or on any of the logs of A. pseudoplatanus. These results are broadly consistent with the relative susceptibility of standing trees of the same tree species at these field sites, although this is the first evidence of "natural" infection of Q. robur by P. ramorum. It is possible that Q. robur trees sometimes become naturally infected without exhibiting external bleeding. Further tests are planned.

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Phytophthora ramorum + P. kernoviae = International Biosecurity Failure

Clive Brasier, Forest Research Agency UK, Forest Research, Alice Holt Lodge, Farnham, Surrey, UK, GU10 4LH; [email protected]

"Sudden Oak Death" caused by Phytophthora ramorum is usually analysed in the context of various scientific disciplines such as pathology, ecology, evolutionary biology, biodiversity, or fire control. However, the "sudden" appearance of this previously unknown, highly damaging and invasive pathogen in North America and Europe, and then soon after the emergence of another invasive Phytophthora, P. kernoviae, tells us that collectively these events are also symptoms of an underlying and more primary issue. This issue is the efficacy - or inefficacy - of international plant biosecurity protocols. If these protocols are not effective, many more "Sudden Oak Deaths" are likely to occur. In that context, as scientists we will continue to study the ripples without being able to prevent the splash. During the first Sudden Oak Death Symposium at Monterey in January 2004 the author presented a scientific health check on the international plant health system governing plant movement. It was concluded that although the international system was well regulated in many countries, e.g. the US and the UK, it could not succeed in protecting our forests and natural ecosystems because it was fundamentally flawed (see The Plantsman 4, 54-57, 2005). One major flaw is that many dangerous pathogens are unknown to science before they invade. Therefore under current plant health protocols it is not possible to take action prior to their arrival because they do not appear on any international quarantine schedules. Indeed both P. ramorum and P. kernoviae were unknown to science before being isolated at disease outbreak sites in the US or Europe. Both pathogens probably arrived on plants imported for the ornamental nursery trade. Both are suggested to have their origin in Asia. The assessment of international plant health protocols presented at the 2004 Monterey Symposium will be updated by reference to the array of new research information now accumulating on the behaviour, population structure, and distribution of P. ramorum and P. kernoviae.

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Population Structure of Phytophthora ramorum in Oregon

Jennifer Britt and Everett Hansen, Oregon State University, Botany and Plant Pathology, 2082 Cordley Hall, Corvallis, OR, USA, 97331; [email protected]; Simone Prospero, INRA Phytophthora ramorum was detected in the forests of southwestern Oregon in 2001. Since then, a major effort to eradicate and prevent the spread of the pathogen has been underway. Previous work has shown that the population of P. ramorum in nurseries in Oregon is genetically distinct from the forest population, suggesting at least two separate introductions into the State. To date, little is known about the method of spread within the forest. Using microsatellite markers, we analyzed the genetic diversity of the P. ramorum population in Oregon forests at several spatial scales from within canker and within single trees, to the entire infested landscape within the Oregon epidemic area. Our goal is to determine if new P. ramorum infections in Oregon forests represent new introductions or result from spread from previous infection sites. Analysis of the distribution of microsatellite genotypes will also give insights to the pathways of disease increase within the infested area.

11

Invasion of Xylem of Mature Tree Stems by Phytophthora ramorum and P. kernoviae

Anna Brown and Clive Brasier, Forest Research Agency, UK, Forest Research, Alice Holt Lodge, Farnham, Surrey, UK, GU10 4LH; [email protected] The aetiology and frequency of Phytophthoras in discoloured xylem tissue beneath phloem lesions was investigated in a range of broadleaved trees infected with P. ramorum, P. kernoviae, and several other Phytophthoras (Plant Pathology 56, in press). Isolation was attempted from the inner surface of 79, 6 x 4 cm sterilised discoloured wood panels from 53 trees. Discolouration mostly extended 1-5 mm into the xylem (76 %) but incursions of 6-10 mm (10 %) and 10-25 mm (14 %) were frequent. Eightyone percent of the wood panels yielded Phytophthora. In 66 cases, both a wood panel and an overlying phloem panel were sampled. In 56 % of these Phytophthora was isolated from both the wood and the phloem panel. In 23 % it was isolated from the wood panel only and in 8 % from the phloem panel only. Small "island" phloem lesions, often in linear arrays adjacent to main lesions, were a common feature of Fagus and Quercus trees infected with P. ramorum or P. kernoviae. Island lesions were often connected by underlying strips or intermittent pits of discoloured xylem in line with the wood grain. P. ramorum, P. kernoviae, and other Phytophthoras were successfully isolated from these connecting xylem features. P. ramorum and P. kernoviae were also recovered from discoloured tissue 5-25 mm below exposed xylem surfaces 12-24 months after the overlying phloem was removed. These results show that Phytophthoras commonly occupy xylem beneath phloem lesions; that they can perennate in xylem tissue; that they can spread in xylem tissue ahead of phloem lesions; and indicate that they may initiate new phloem lesions in this way. Such colonisation must lead to at least local xylem dysfunction. It is recommended that, if xylem discoloration is present, isolation of Phytophthora should be attempted from the xylem as well as the bark. Also, that removal of infected outer sapwood should be undertaken during excision of Phytophthora bleeding lesions for disease control and in protocols aimed at preventing national or international spread of tree stem Phytophthoras. 12

Recovery of Phytophthora ramorum from Mistletoe and California Bay Inflorescences and Possible Implications Relating to Disease Spread

Gary A Chastagner, Kathy Riley, and Norm Dart, Washington State University, Puyallup, WA, USA, 98371; [email protected]

Since 2005, we have been studying the spread and development of Phytophthora ramorum at a Christmas tree farm near Los Gatos, CA. This research has shown that distance from infected plants, predominantly California bay laurel (Umbellularia californica), is an important factor relating to the infection of Douglas-fir (Pseudotsuga menziesii) and grand fir (Abies grandis) Christmas trees at our research site. In a few instances, we have observed the development of a sunken pitchy canker on the older grand fir branches. These cankers eventually girdle the branch, resulting in a branch flagging. To understand the origin of these cankers, we have been trying to determine if the pathogen is spreading down the branch from infected shoot tips, if it is spreading from infected small interior secondary shoots near the canker, or if there are some conditions that allow for direct infection of the older needles or the bark. Trees that develop this branch canker tend to have high levels of shoot infection and often have detached bay leaves and inflorescences lodged among the older needles on the branches. Because flowers can serve as a source of inoculum for some pathogens, we have looked into the possibility that the detached bay inflorescences may be infected by P. ramorum. We have been able to isolate P. ramorum from inflorescences still attached to bay trees, but not on the detached flowers on any of the conifers. Thus, additional work is needed to determine if infected bay flowers play any role in the development of the pitchy cankers. In 2005, a few white fir (A. concolor) Christmas trees were found to have a limited number of P. ramorum infected shoots at another farm near our research site. The infection on these trees was unexpected because they were not adjacent to known hosts of P. ramorum. Some of the infected trees were beneath a large black walnut (Juglans nigra) tree that was infected with mistletoe (Phoradendron serotinum subsp. macrophyllum). In 2006, we collected a number of pieces of mistletoe that had fallen out of the walnut tree. Although no P. ramorum was recovered from any of the mistletoe leaf or stem tissue, it was isolated from a blackened inflorescence. Work is currently in progress to confirm that mistletoe plants in the tree are infected and determine what role they may play in the spread of disease to the white fir.

13

Spread and Development of Phytophthora ramorum in a California Christmas Tree Farm

Gary Chastagner, Kathy Riley, and Norman Dart,Washington State University, Research and Extension Center, 7612 Pioneer Way East, Puyallup, WA, USA, 98371; [email protected] Since 2005, the spread and development of Phytophthora ramorum has been monitored in a 23-acre U-cut Christmas tree farm near Los Gatos, CA. Conifers being grown at this site include Douglas-fir (Pseudotsuga menziesii), grand fir (Abies grandis), giant sequoia (Sequoiadendron giganteum), Scots pine (Pinus sylvestris), white fir (A. concolor), and California red fir (A. magnifica). Some known P. ramorum hosts in the infected forest adjacent to the edge of the Christmas tree farm include: California bay laurel (Umbellularia californica), madrone (Arbutus menziesii), big leaf maple (Acer macrophyllum), false solomon seal (Maiantheumum racemosum), toyon (Heteromeles arbutifolia), coast redwood (Sequoia sempervirens), and tanoak (Lithocarpus densiflorus). After mapping the perimeter of the farm to identify areas where Ramorum Blight was evident, 500 trees in the largest area with a past history of infection were mapped, tagged, and measured for height. A series of transects were established from the edge of the forest into the Christmas trees in this area to monitor the spread of P. ramorum from infected plants (predominantly bay) along the edge of the forest into the Christmas tree plantation. The length of these transects ranged from 17 to 27 m. Container-grown Douglas-fir and grand fir seedlings that had just broken bud and small rhododendron plants were also placed along three transects at approximately 0, 3.5, 8, and 13 m from the forest edge. Periodically during the spring and summer, the level of infection and extent of shoot dieback has been assessed on the tagged trees and containerized seedlings. In both 2005 and 2006, new shoot infections on the Christmas trees only developed in the spring and initial dieback symptoms were limited to newly expanded shoot tips. Environmental conditions during spring 2005 were much more favorable to initial shoot tip infections than in 2006. In particular, along the transects where grand fir were underneath the canopy of infected bay, virtually all of the new shoots were infected shortly after bud break in 2005. The progression of dieback on infected shoots of Douglas-fir and grand fir in 2005 progressed for about 4 weeks after 14

the initial appearance of symptoms, typically spreading about two inches into the previous year's growth. The extent of dieback did not increase between early summer and mid-November. Infection rates and disease severity were also much higher among the seedlings that were placed along the interface of the forest and Christmas tree farm in 2005. On May 19, 2005, 81.7 and 94.3 % of the Douglas-fir and grand fir seedlings, respectively, that had been exposed since April 21 had been infected. The percentage of each seedling that was killed as the result of shoot dieback averaged 52.8 % for the Douglas-fir and 81.2 % for the grand fir. In 2006, infection of conifer seedlings and rhododendrons only occurred during exposure periods when precipitation occurred. The infection of Douglas-fir and grand fir trees and seedlings along the transects indicate that distance from infected plants (predominantly bay) within the forest is an important factor relating to the infection of the Christmas trees. Most of the infected Christmas trees and seedlings occurred within 2 to 4.4 m of the edge of the forest. Virtually no infection was evident on Christmas trees that were 5 to 8 m away from the forest edge.

15

Detection and Quantification of mRNA by Reverse Transcription Real-Time PCR as an Indicator of Viability in Phytophthora ramorum Infected Soil and Plant Material

Antonio Chimento and Matteo Garbelotto, UC Berkeley, Department of Environmental Science, Policy, and Management, 137 Mulford Hall, Berkeley, CA, USA, 94720-3114; [email protected] Real-Time PCR technologies offer increasing opportunities to detect and study phytopathogenic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing both real-time analysis of the reaction kinetics and quantification of specific DNA targets. Before the development of Real-Time PCR and the opportunity to provide quantitative data, the risk of false-positive PCR results due to detection of dead cells was considered only a minor setback. This, therefore, has led to a renewed interest in the risk of false-positive PCR results. In order to deal with risks of false-positive results, we developed a new RT-PCR assay based on the use of mRNA as a viability marker, on the basis of its rapid degradation compared to DNA. We developed new primers, specific for P. ramorum, designed in the cytochrome oxidase gene encoding subunits I (COXI). To evaluate the specificity of the method, 4 isolates of P. ramorum and 11 different Phytophthora species were tested. Sixty symptomatic bay leaves from 3 different sites in California and sixty symptomatic bay leaves collected in 3 different seasons of the year from China Camp State Park, were collected and plated on PARP selective media for Phytophthora and tested with the new RT-PCR method and compared with a TaqMan and SybrGreen Real-Time PCR assay after a classical DNA extraction. Results showed that after 9 days RNA of freeze-dried killed P. ramorum was undetectable while DNA gave a positive signal. Furthermore, data from the new assay were more correlated to the results obtained after isolation on selective media whereas DNA-based results showed more positive samples. This indicates that by using the new RT-PCR method, the risk of false-positive PCR results due to detection of dead cells can be minimized.

16

Stand Level Infection and Mortality Dynamics in Phytophthora ramorum Infested Redwood-Tanoak Forests: Patterns and Predictions Based on Five Years of Monitoring

Richard C. Cobb, UC Davis Graduate Group in Ecology, Plant Pathology Department, One Shields Ave, Davis, CA, USA, 95616; [email protected]; Shannon Lynch and David M. Rizzo, UC Davis; Ross K. Meentemeyer, University of North Carolina at Charlotte

Management efforts in Phytophthora ramorum infested forests are currently operating without empirically defined rates of mortality and infection. To develop long-term systematic management plans for California forests we should have a strong modeling framework that allows us to predict the spread and intensification of the pathogen on individual sites, target the most effective control treatments, and evaluate the effectiveness of these treatments. To date, most modeling approaches have focused on the landscape and national levels to determine at risk areas and inform regional planning and quarantine efforts. This study will examine the rates and patterns of P. ramorum infection in redwood-tanoak forests to evaluate temporal patterns and interactive effects of vegetation structure within stands. Disease incidence and tree mortality in redwood-tanoak forests were determined by repeated sampling across a system of 120 plots at five long-term research sites from 2001 through 2006. Plots were located within the known geographic area of P. ramorum in California and ranged from Monterey to Sonoma counties. Data were fit to a SI (susceptible infected) disease dynamics model parameterized with plot level data. Effect of year-to-year climate variation was evaluated by comparing year-to-year infection response to a linear null model. Disease progression significantly departed from the null model in bay laurel (Umbellularia californica) and tanoak (Lithocarpus densiflorus) suggesting weather and abundance of susceptible species are important in year-to-year pathogen increases. Redwood (Sequoia sempervirens) showed linear rates of increase and overall lower levels and slower rates of infection compared to bay laurel and tanoak. Mortality levels were significant for tanoak but not bay laurel or redwood over the five year period. Further analysis will use statistical modeling and validation to evaluate the role of vegetation structure on rates of infection and mortality of tanoak. This effort will help focus stand-level intervention by identifying local areas where control strategies will be most effective.

17

Landscape Connectivity Influences the Establishment of Phytophthora ramorum

T. Emiko Condeso, Sonoma State University, 899 Norlee St., Sebastopol, CA, USA, 95472; [email protected]; Ross K. Meentemeyer, University of North Carolina at Charlotte Plant pathogen invasions are inherently spatial, but few studies have demonstrated the role of landscape connectivity in establishment and spread. In this study, we investigated two questions to evaluate the effect of the spatial pattern of host habitat on the establishment and spread of Phytophthora ramorum, causal agent of Sudden Oak Death: (1) does the spatial pattern of host habitat predict P. ramorum disease severity, and is this relationship scale-dependent; and (2) what influence does spatial pattern have on the optimal microclimate conditions for P. ramorum reproduction? To examine these questions, we mapped the spatial distribution of suitable forest habitat for P. ramorum and established 86 randomly located field plots within a 20 km2 region of Sonoma County, CA. For each plot, we quantified P. ramorum disease severity and measured the abundance of woody species. Disease severity in each plot was examined in relation to the amount of surrounding host habitat measured for nested landscapes of increasing scale. P. ramorum disease severity was greatest in plots surrounded by a high proportion of contiguous forest, after accounting for plot-level variables of host abundance, elevation, canopy cover, and microclimate. The explanatory power of the model increased with increasing scale up to 200 m, but was not significant at scales less than 50 m. High disease severity was associated with cooler temperatures in the field rather than the lab determined, optimal range for pathogen reproduction. Variation in microclimate conditions was explained primarily by elevation, not microclimate-related edge effects, indicating that disease severity is influenced by the connectivity of host vegetation. Both landscape-scale configuration and local composition of host habitat are related to the severity of this destructive forest disease. Increased disease severity within contiguous woodlands may have a considerable impact on the composition of such woodlands, with cascading effects on the population dynamics of both host and pathogen. 18

Human Activity and the Spread of Phytophthora ramorum

J. Hall Cushman, Sonoma State University, Department of Biology, Rohnert Park, CA, USA, 94928; [email protected]; Michelle Cooper, Bodega Marine Laboratory, UC Davis; Ross Meentemeyer, University of North Carolina - Charlotte; Shelly Benson, USDA Forest Service - Olympic National Forest Increasing numbers of studies are finding that humans can facilitate the spread of exotic plant species in protected wildlands. Hiking trails commonly serve as conduits for invaders and the number of exotic plant species occurring in protected areas is often correlated positively with visitation rates. Despite such evidence linking human activity to the spread of exotic plants, few studies have addressed this possibility for plant pathogens. Over the past four years, we have been evaluating the role that humans play in promoting the spread of P. ramorum and the disease it causes. Our previous research has suggested that human activity is hastening the spread of P. ramorum in northern California's Sonoma County: the pathogen was more commonly found in soil on hiking trails than from soil in adjacent areas off trails; public lands open to recreation had higher proportions of diseased host trees than private lands; and the chance that host trees were infected by P. ramorum increased as the density of human populations increased in the surrounding area. Collectively, these data suggest that human activity can inadvertently disperse P. ramorum throughout the landscape, further spreading the pathogen into already infected areas and introducing it into previously uninfected areas. More recently, we have conducted additional studies that further link two forms of human activity - hiking and mountain biking - to the dispersal of P. ramorum. First, at a nature preserve in Sonoma County, we have shown that hikers can disperse P. ramorum in soil on their shoes at least 60-100 m into areas that lack local sources of inoculum. Second, we found that 5-10 % of the visitors entering a recreational area in Marin County had the pathogen in soil on their shoes and tires, and 20-30 % carried it out with them. Although hikers and mountain bikers did not differ significantly in the capacity to transport P. ramorum, there was a trend indicating that during dryer conditions, the further a person 19

traveled along a trail, the more likely they were to pick up and transport the pathogen. In addition, although our data suggest that humans can serve as effective dispersal agents, the temporal window for doing so is constrained, as the pathogen could not be cultured from soil on hikers' shoes after 24 hours, although this time was extended to at least 72 hours if the soil on hiking shoes was kept moist. These results suggest that human dispersal of P. ramorum may be limited to certain kinds of situations: further spread of the pathogen in already infected areas or instances in which visitors move rapidly from one region to another, especially when hiking shoes or mountain bikes have been stored in moist conditions. In summary, our research suggests that there may be conflicts between human activities and disease spread, and that efforts to address this epidemic may require aggressive management, which may be logistically and politically challenging to implement.

20

Effect of Environmental and Seasonal Factors on the Susceptibility of Different Rhododendron Species and Hybrids to Phytophthora ramorum

Isabelle De Dobbelaere, Kurt Heungens, and Martine Maes, ILVO - Unit Crop Protection, Burg. Van Gansberghelaan 96 bus 2, Merelbeke, Belgium, B-9820; [email protected] Commercial Rhododendron plants are the most important hosts of Phytophthora ramorum in Europe. Plant health inspection services and growers would benefit from information on the relative susceptibility of different Rhododendron species and hybrids. In order to provide that information we evaluated screening methods and started screening 63 Rhododendron species and hybrids in 2004. The results showed significant differences in susceptibility between the Rhododendron hosts and were presented at the SODII Symposium. However, results indicated that the most informative screening method, the one involving immersion of detached leaves in a zoospore suspension, resulted in variable results depending on environmental factors and the time of year the assay was conducted. Because of this variation in susceptibility within a cultivar, the study was extended in 2005 and 2006. This included re-testing many cultivars and the direct analysis of some time and environmental factors on symptom development. A total of 80 Rhododendron hosts (24 species and 56 hybrids) were screened during July to September of 2004, 2005, and 2006. Species were selected to represent the main subdivisions within the genus Rhododendron. Hybrids were selected based on their economic importance. The assays were performed in batches and included the same control cultivar in each batch. Two screening methods were used. A method involving non-wounded leaves was used to estimate the ability of the hosts to resist tissue penetration. A method involving wounded leaves was geared at evaluating the resistance to pathogen growth inside leaf tissue. In contrast with the method with wounded leaves, the method with non-wounded leaves revealed a considerable difference in level of resistance between cultivars or species. The average level of susceptibility was significantly different between the years. There was also a seasonal variation in susceptibility. For this experiment, leaves of the control cultivar were collected and inoculated on a bi-weekly basis during a period of two years. Leaf age also played an 21

important role in susceptibility tests. This effect was species dependent and linked to morphological features of the leaves. Environmental factors that affect stomatal regulation, such as temperature at the time of leaf collection, also seemed to have an effect on the degree of symptom development. Taken together, this information leads to more reliable classification of the susceptibility of Rhododendron (and possibly other) hosts to P. ramorum.

22

Sporulation of Phytophthora ramorum and P. kernoviae on Asymptomatic Foliage

Sandra Denman, S. A. Kirk, E. Orton, and J. F. Webber, Forest Research Station, Alice Holt Lodge, Farnham, Surrey, England, GU10 4LH; [email protected]; E. Moralejo, Instituto Mediterraneo de Estudios Avanzados P. ramorum and P. kernoviae are newly discovered invasive Phytophthoras affecting a wide range of ornamental trees and shrubs and forest species. The diseases they cause include leaf necrosis, shoot tip dieback and bleeding cankers on tree trunks resulting in the death of some infected trees. Both pathogens are now present in south-west England. P. ramorum also occurs in Europe and is prevalent in California and parts of Oregon where it causes a lethal disease of native oaks, commonly referred to as Sudden Oak Death, as well as causing damage to ornamental plants such as Camellia and Rhododendron. P. kernoviae previously only reported from the UK has now just been discovered in New Zealand. The epidemiology of both pathogens is similar but complex; sporulation occurs on infected shoots and foliage but not on bleeding stem cankers. Foliar hosts are thus a crucial component of disease epidemiology because they act as the platforms from which inoculum is dispersed and are the drivers of epidemics. During the evaluation of field trials established to assess inoculum density and natural infection, we discovered by logical deduction that naturally infected, asymptomatic leaves support sporulation of both pathogens. Laboratory trials were then carried out to investigate whether artificially-inoculated, detached, asymptomatic foliage and fruit also supported sporulation. Asymptomatic leaves of 63 % of trap plants yielded P. kernoviae and 31 % of trap plants yielded P. ramorum. In this paper we discuss possible reasons for this behavioural mode as well as the implications for disease spread.

23

Genotyping Indicates Nursery and Early Phytophthora ramorum Collections are Indistinguishable, while Showing Current Local Diversification of Wild Populations Driven by Geography

Matteo Garbelotto, AnnaMaria Vettraino, and Noah Rosenzweig, UC Berkeley, Department of Environmental Science, Policy, and Management, 137 Mulford Hall, Berkeley, CA, USA, 94720-3114; [email protected] We were able to unequivocally identify hundreds of P. ramorum isolates from a large number of sites and from nurseries as belonging to approximately 40 different genotypes. The study included both isolates from `historical' pathogen collections obtained until 2002, and from collections obtained in 2005. A contingency analysis of genotype diversity indicates a significant overlap between nursery isolates and historical collections. On the contrary, current collections from different sites show very little overlap and highlight ongoing differentiation among populations of the Sudden Oak Death pathogen. In light of the recently proven strict clonality of this organism in the wild, we infer that mutations and mitotic recombination events are responsible for the increase in genotypic diversity and significant genetic differentiation among populations found in different sites. Population differentiation may lead to subgroups of the pathogen characterized by different pathogenicity traits, and thus, care should be given to avoid movement of isolates from one area to another. Significant overlap between historical wild collections and nursery isolates indicates an early exchange of pathogen genotypes between the wild and commercial nurseries.

24

Vegetation Response Following Phytophthora ramorum Eradication Treatments in Southwest Oregon Forests

Ellen Goheen, USDA Forest Service, 2606 Old Stage Road, Central Point, OR, USA, 97502; [email protected]; Everett Hansen, Oregon State University; Alan Kanaskie, Oregon Department of Forestry Sudden Oak Death, caused by Phytophthora ramorum, was identified in late July 2001 in forested stands on the Southwest Coast of Oregon in Curry County where it was killing tanoak (Lithocarpus densiflorus) and infecting Pacific rhododendron (Rhododendron macrophyllum) and evergreen huckleberry (Vaccinium ovatum). Most stands in this area have an overstory component comprised predominantly of tanoak and Douglas-fir (Pseudotsuga menziesii), with lesser amounts of red alder (Alnus rubra), Oregon myrtle (Umbellularia californica), Cascara (Frangula purshiana), Pacific madrone (Arbutus menziesii), and bigleaf maple (Acer macrophyllum). The understory species mix includes substantial amounts of tanoak, Pacific rhododendron, Oregon myrtle, and evergreen huckleberry, as well as a number of other species. Many of the species in both the overstory and understory produce prolific stump sprouts following cutting or burning. Treatments to eradicate the pathogen from affected sites began in the fall of 2001 and consisted at that time of cutting, piling, and burning all infected host vegetation and any known Oregon host species within a 50 to 100 foot buffer of infected plants. Patch size of treatment areas ranged from 0.5 to 11 acres. Since that time, additional disease centers have been identified and eradication treatments have been completed at every site. Some treatments have been adjacent to those sites treated initially while others are distinct centers. Over the last five years, treatment methods have been altered to reflect increased understanding of host susceptibility and pathogen survival and spread. Additional treatment components have included various combinations of backpack spraying herbicides to kill stump sprouts, stump-top treatments with herbicides to prevent tanoak sprouting, injecting all tanoaks greater than 1 inch diameter with herbicides to prevent sprouting, raking, piling, and burning all Oregon host material, and increasing buffer width to 300 feet. Some sites have been planted with conifer seedlings while others have not. 25

There is interest in understanding how site vegetation responds to treatment, how treatments affect the relative susceptibility of host species, and how the impacts of the treatments themselves influence changes in forest structure and species composition. Data on vegetation response will be presented from permanent monitoring plots that reflect vegetation changes at the stand-level, stump-based plots that follow known vegetation changes around known infected trees, and vegetation transects across treated areas that quantify ingrowth, sprouting and relative host and nonhost abundance.

26

What can Availability of the Phytophthora ramorum Genome do for us?

Niklaus J. Grünwald, Horticultural Crops Research Laboratory, USDA ARS, 3420 NW Orchard Ave., Corvallis, OR, USA, 97330; [email protected] The complete genomes of Phytophthora ramorum and P. sojae were sequenced in 2004. One obvious question arising is what contributions the availability of a genome sequence has for understanding the biology of Phytophthora spp. and what the implications for management of Sudden Oak Death in the field might be. Sequencing of two Phytophthora genomes right away allowed for direct comparison for similarities and differences between these two organisms. Of the 19,027 predicted genes in P. sojae and 15,743 gene models in P. ramorum, 9,768 are predicted to have the same function. These two genomes both revealed a rapid expansion and diversification of many proteins families associated with plant infection including different classes of pathogen effectors. Pathogen effectors can serve a virulence function on behalf of the pathogen or trigger a rapid defense response in resistant hosts. Two protein motifs (RxLR and dEER) are shared by the four known effectors in plant pathogenic oomycetes. Genome anlayses identified a diverse superfamily of approximately 350 genes in P. ramorum that share these motifs. These have been termed avirulence homolog genes. With these sequences in hand we were able to clone homologous Avh loci from sister-taxa P. lateralis and P. hibernalis. Availability of the P. ramorum genome sequence has also resulted in several practical applications. We have for instance started to investigate which genes are differentially expressed during different life-stages (mycelium, zoospores, etc.) and during plant infection using microarrays developed from the current genome annotation. The genome has been mined for molecular loci that can be used for pathogen identification and genotyping. The genomes have been helpful in selecting sequence loci that can be used for constructing a phylogeny of the genus Phytophthora (http://www.phytophthoradb.org/). The genome has also been helpful in finding simple sequence repeats and sequence loci that have been used to study the population structure and migration of clones. Availability of the genome sequences has already provided advances and a new understanding of Phytophthora biology. Many promising new discoveries are sure to follow. 27

Linking Sudden Oak Death Risk Models with Economic Impact Assessments

Thomas Holmes and Bill Smith, USDA Forest Service, Southern Research Station, Forestry Sciences Lab, PO Box 12254, Research Triangle Park, NC, USA, 27709; [email protected] Sudden Oak Death (SOD) was first reported in California in 1995. Since then, tens of thousands of tanoaks, coast live oaks, and California black oaks have been killed. SOD has the potential to infect extensive forest areas in the eastern US containing Northern red, Southern red, and pin oaks. In 2003, an infected nursery in Southern California shipped over 66,000 plants to the Southeastern and Mid-Atlantic states. Although little is known of the location of infected plants sold prior to inspection, voluntary reporting by 166 residential landowners in Georgia showed that 1.4 % of purchased plants were infected. To assist in monitoring and management activities, SOD risk models have been built and are being used to guide prevention activities in California, Oregon, and the Eastern US. SOD risk models span a range of methods from relatively simple rule-based models to more complex models based on various statistical techniques, and different models make different predictions of relative risk across spatial units. Because SOD risk models rely on a spatial representation of risk and use GIS tools, they can form the basis for linking economic assessments of actual and potential economic impacts. Linking SOD risk models with actual economic impacts and economic values at risk can improve risk assessment and decision analysis and help prioritize management efforts. Market and non-market valuation methods have been applied to the problem of estimating economic losses from forest disturbances. Market models have been used to estimate impacts on timber producers and consumers from disturbances such as insect epidemics, hurricanes, and wildfires. Non-market valuation studies have employed a variety of tools such as contingent valuation, travel cost, and hedonic property value methods to estimate the economic impacts of exotic insects and wildfires on consumers of forest services. These studies demonstrate that forest disturbances can have large

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