Phytophthora Species and Riparian Alder Tree Damage in Western Oregon

Home , Alniphagus, Alnus cordata, Alnus rhombifolia, Alnus rubra, Eriocampa ovata, Notholithocarpus

AN ABSTRACT OF THE DISSERTATION OF

Laura L. Sims for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on February 13, 2014.

Title: Phytophthora Species and Riparian Alder Tree Damage in Western Oregon

Abstract approved:

Everett M. Hansen

The genus Phytophthora contains some of the most destructive pathogens of forest trees, including the most destructive pathogen of alder in recent times,

Phytophthora alni. Alder trees were reported to be suffering from canopy dieback in riparian ecosystems in western Oregon, which prompted a survey of alder health and monitoring for P. alni. In 2010 surveys in western Oregon riparian ecosystems were initiated to gather baseline data on damage and on the Phytophthora species associated with alder. Damage was recorded and analyzed from transects containing alder trees with canopy dieback symptoms according to damage type: (1) pathogen, (2) insect, or

(3) wound. Phytophthora species from western Oregon riparian ecosystems were systematically sampled, isolated, identified, stored and compared. Koch’s Postulates were evaluated for three key Phytophthora species recovered: P. alni, P. siskiyouensis and P. taxon Oaksoil, and alder disease in the western United States was described.

Then, the ecological role of the most abundant Phytophthora species from streams was evaluated. The data indicated that many of the same agents reported causing damage to alder trees in the western United States were also damaging alder trees in western

Oregon including the alder flea beetle, sawflies, flood debris, Septoria alnifolia, and

Mycopappus alni. The most important damage correlated with canopy dieback was incidence of Phytophthora cankers, and isolation of Phytophthora siskiyouensis. In the initial systematic survey of Phytophthora species, 1190 individual Phytophthora isolates were recovered but were of many different species. In the survey of alder roots, P. alni subsp. uniformis was one of the species recovered from necrotic red alder roots, but overall incidence was low; it was isolated four times. From the evaluation of Koch’s postulates, Phytophthora canker of alder in the western United States was described, and is a bole canker caused by Phytophthora. Phytophthora canker of alder was only found caused by P. siskiyouensis in nature, and it was isolated 74 times. Isolation was mainly from bole cankers and diseased roots on red and white alder, and from water and alder leaf debris floating in the stream. The most abundant Phytophthora species associated with red alder is an informally described species P. taxon Oaksoil, which appears to be a relatively benign aquatic saprotroph of alder leaf debris. Canopy dieback was more prevalent in riparian alder trees from transects with P. siskiyouensis than from transects with P. taxon Oaksoil but without P. siskiyouensis (70% and 35%, respectively).

The informally described P. taxon Oaksoil from western Oregon is formally described here as P. obrutafolium sp. nov., closely related to P. bilorbang from western Australia, and P. taxon Oaksoil ss from an oak forest in France. In summary, other agents besides

Phytophthora can damage alder trees in western Oregon. Many Phytophthora species associate with alder in western Oregon but not all of them are important damaging agents of alder. However, Phytophthora canker of alder is widespread in western

Oregon. In the United States, Phytophthora canker of alder has only been found to be caused by P. siskiyouensis.

Phytophthora Species and Riparian Alder Tree Damage in Western Oregon

by Laura L. Sims

A DISSERTATION

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Presented February 13, 2014 Commencement June 2014

Doctor of Philosophy dissertation of Laura L. Sims presented on February 13, 2014.

APPROVED:

Major Professor, representing Botany and Plant Pathology

Head of the Department of Botany and Plant Pathology

Dean of the Graduate School

I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request.

Laura L. Sims, Author

ACKNOWLEDGEMENTS

I sincerely thank my advisor Everett Hansen. I truly appreciate his facilitation of my creative drive and tolerance of my unerring biological curiosity in regards to pathology. I appreciate the time I was allowed to spend on Mary’s Peak with Toby

Child’s Poria weirii my first and last summer while obtaining my degree. I would also like to thank both him and Barb for inviting my family to stay in their home, twice.

I sincerely thank Ellen Goheen and her U. S. Forest Service crew, which contended with the most difficult terrain of the alder project. I truly appreciate the guidance, cooperation, and direction she gave to the project from the beginning.

I truly thank Alan Kanaskie, Michael Thompson, and Jon Laine from the Oregon

Department of Forestry for their excellence in fieldwork and for their time. I could not have completed my dissertation project without their assistance. I would also like to thank Mike McWilliams for his enthusiasm and advice about forest pathology.

I truly thank Wendy Sutton for her help in processing the seemingly endless stream of samples I brought into the lab for a while. The lab is a very comfortable place to work because of Wendy’s laugh and her great organization. I would also like to thank

Paul Reeser for his time, for his patience, and for showing me the art of seeing

Phytophthora amongst the myriad of life that I dragged back from the forest.

I am grateful to the Oregon State University Botany Department for being a welcoming place while I completed my degree. I truly appreciate the committee that granted me the Anita Summers Fund, which provided a large part of the funding for a work trip conference to Spain.

I thank my family for engraining in me the importance of hard work and perseverance. To my sister and brother, Amy and Joe that both accomplished so much while I was completing my degree. To my “Gunny” Sergeant Grandfather, Joe

Whealdon, who passed on while I was working on my dissertation, I miss you. Thank you, to the hard working women in my family especially my mother Elisa and my aunt

Dawn, who inspired me to stay strong and focused.

I would like to thank the Forest Health Monitoring Program of the USDA Forest

Service, Pacific Northwest Region, which generously supported survey efforts. I truly appreciate all the financial support for the project, and the opportunities to attend and communicate about my progress at conferences. Particularly I appreciate the opportunity to attend the Western International Forest Disease Work Conference

Meetings and the International Union of Forest Research Organization Meeting. I was able to meet and communicate with the forest pathology family. I pray they will keep me.

TABLE OF CONTENTS

Page CHAPTER 1: Dissertation Introduction …………………………………...... 1

The Genus Phytophthora …………………...... ……………………….……………………....2

Alder…………...... ……………...7

Alder dieback in Oregon ...... 9

Dissertation Objectives ..………...... 10

References…..…………………………………………………..…………………………………..…….12

CHAPTER 2: Phytophthora siskiyouensis and other damaging agents of alder trees in western Oregon riparian ecosystems ...…...... ……………19

Introduction………………………….……………………………..……...…...…………....…………19

Pathogens……………………………………………………………....…..……………………….24

Insects……………………………………………………………………….………………………….26

Wounds………………………………………………………………………….…………………….28

Materials and Methods…………...……...………….……………...... 29

Transects……………………………….…………………………..………………...... ……………29

Data Collection…….……..………………………………………………………...... 31

Data Analysis……….….……………………..……………………………………...... 33

Results………………………………………………..…………..…………………………...... 36

Canopy Dieback…..…………………………..…………………………………...... 36

Pathogens……………..…………………..…………………………………………...... 38

TABLE OF CONTENTS (Continued)

Page Insects…………………………..………………………………………………………...... 46

Wounds…………….……………………..…………………………………………...... 47

Discussion...... 49

Canopy Dieback...... 50

Pathogens...... 50

Insects...... 52

Wounds...... 53

Other...... 54

Conclusions……..…………….……………………..…………………………………………………...56

References…………………………….…………………………..……………………………………....57

Chapter 3: Phytophthora species from riparian alder ecosystems in western Oregon, USA…..………………………………..………………………………………………………………………………………..63

Introduction……………..…………………..……………………..…………………………………….63

Materials and methods…………………………..………………………………………….……….72

The assemblage of Phytophthora species………..………………………….………..72

Alder survey location and layout..………………...…………………….………..72

Sampling Phytophthora species from stream water and the rhizosphere……………………………………………………………………………………73

Coastal subset survey location……………………………………………………....75

Sampling Phytophthora species from alder roots…………………………..76

TABLE OF CONTENTS (Continued)

Page Phytophthora species determination…………....…………...... 76

Data analysis……………….……………………………………………...... 78

Results……………………………………………………………………………………………...... 80

The assemblage of Phytophthora species……………………………...... 80

The overall assemblage……………………………………………………...... 80

Incidence of Phytophthora alni, and location details…………...... 80

Phytophthora species assemblage from stream water and the rhizosphere………………………………………………………………...... 82

Comparison of Phytophthora from streams and the rhizosphere…………………………………………………………………...... 87

Comparing species and clade composition from fine and woody roots…………………………………………………………………………...... 91

Discussion………………………………………………………………………...... 92

The assemblage of Phytophthora species…………………...... 92

The overall assemblage…………………………………………...... 92

Evaluation for P. alni……………………………………………...... 94

Comparing situations…………………………………………………...... 94

Other species………………………………………………………………...... 95

Comparing species and clade composition from stream water and the rhizosphere……………………………………………………...... 101

TABLE OF CONTENTS (Continued)

Page Comparing species and clade composition from fine and woody roots………………………………………………………………...... 102

Molecular methods……………………………………………………...... 103

Conclusions…………………………………………………………………….……...... 106

References…………...………………..…………………………...... 107

CHAPTER 4: Pathogenicity of Phytophthora siskiyouensis, P. alni subsp. uniformis and P. taxon Oaksoil to alder trees in western Oregon…………….…...... 114

Introduction………………………………………………………………………...... …...... 114

Materials and Methods……………………………………………...... 124

Survey Efforts…………………………………...... 124

Sampling Efforts………………………………...... 125

Phytophthora species determination…………………...... ………...... 126

Alder for the inoculation trial……………………………...... ………...... …..128

Stem inoculation trial…………………………………………………...... 128

Modeling a treatment effect………………………………...... …...... ……131

Results…………………………………………………………………...... …...... ………….135

Correlating canopy dieback with Phytophthora species...... 138

Pathogenicity test results from the model…………...... ………...... ………140

Discussion………………………………………………………………...... ……………………..142

Phytophthora canker of alder……………………………...... ………………….….142

TABLE OF CONTENTS (Continued)

Page References………………………………………………..……………...... …………………...... ….148

Chapter 5. Phytophthora obrutafolium species nov., an Alnus rubra leaf saprotroph closely related to P. bilorbang and P. taxon Oaksoil ss...... 152

Introduction……………………………………………………………..…………...... …...... 152

Methods...... 156

Survey...... 156

Physiology...... 160

Morphology...... 163

Phylogeny...... 165

Ecology...... 166

Results...... 173

Physiology...... 173

Taxonomy...... 176

Description...... 176

Phylogeny...... 181

Ecology...... 182

Discussion...... 187

Conclusions...... 190

References...... 191

Chapter 6: Dissertation Conclusions...... 194

Motivation...... 194

Dissertation goal and objectives...... 195

TABLE OF CONTENTS (Continued)

Page Chapter 2...... 196

Chapter 3...... 197

Chapter 4...... 198

Chapter 5...... 199

Summary...... 200

References...... 201

Bibliography...... 202

LIST OF FIGURES

Figure Page

2.1 In western Oregon riparian ecosystems red alder trees with canopy dieback .....20

2.2 Locations of transects in western Oregon riparian ecosystems for the alder survey. Latitude and longitude of the southernmost transect (224): N° 42.0060, W° 124.2132.…………………………………………………………...……………...……...... 30

2.3 A red alder tree with recent canopy dieback in western Oregon riparian ecosystems. Note: A standing dead alder tree is pictured left of the tree with canopy dieback …….....…...…………………………………………………………………………...... 37

2.4 Common foliar damage of alder in western Oregon (Left to right from top) - Ascomycete leaf spot pathogen Septoria alnifolia was recovered from alder trees with diseased leaves examined during the survey, clusters of pycnidia, were in the center of leaf spots. - Brown leaf blotch on a red alder leaf collected during the survey, note the white conidiomata of the ascomycete leaf spot pathogen Mycopappus alni. - Brown leaf blotch whole leaf symptom from a red alder trees with diseased leaves, the white spots are signs of the pathogen. - The most commonly observed insect damage of alder trees was from insects like the alder flea beetle Altica ambiens (larval stage). - Sawflies larva were common on alder leaves, the woolly alder sawfly larvae (Eriocampa ovata) feeding on an alder leaf. Alder leaf damaged from feeding by sawfly larvae. White bar = 5cm. Black bar = 1 cm...... …………………………………...... 40

2.5 Phytophthora cankers on alder trees in western Oregon riparian ecosystems. (Left) Alder tree with Phytophthora type canker (SC1) in an alder stand. Phytophthora siskiyouensis was recovered from the canker. The large bleeding canker extended up the bole well over 1.5 m in length with small bleeding cankers continuing to at least 2.5 m (arrows). (Top right) Alder tree with SC1 in an alder stand (chisel is for scale the blade is 3.7 cm wide). (Bottom right) SC1 ooze on the outside of the bark discolors the outer bark orange, red and dark grey; cankers were sometimes accompanied by dry cracked bark...... 44

3.1 Phytophthora species composition from (a) stream water and (b) the rhizosphere associated with alder in western Oregon riparian ecosystems. Recovery was from 88 transects.………….....…...... …...... 86

LIST OF FIGURES (Continued)

Figure Page

3.2 Phytophthora species composition from (a) fine roots and (b) woody roots of red alder from western Oregon riparian ecosystems. Recovery was from 18 transects. …………………...... …...……………………………………………………………………...... 89

3.3 Diseased alder root piece that recovered Phytophthora species from both fine and woody root necrosis. (F) fine root necrosis (W) woody root necrosis...... 90

3.4 P. europaea from western Oregon with amphigynous attachment of the antheridium to the oogonium stalk. Bar 50 μ …………..………….....….…...…...... 101

4.1 Phytophthora bole cankers on alder infected with P. siskiyouensis in western Oregon riparian ecosystems...... …...... 119

4.2 Alder leaf debris accumulates in streams...... 121

4.3 Normal quantile-quantile plot of residuals from the model before (a) and after (b) applying a natural log transformation to canker length...... …...... 133

4.4. From left to right cankers resulting from inoculation with: a carrot agar plug, P. taxon Oaksoil, P. siskiyouensis, and P. alni subsp. uniformis. Each alder tree unit pictured was stem wound inoculated over a pinprick wound. Trees were incubated in the greenhouse for 15 days prior to examining cankers...... 141

4.5 Symptoms produced by P. siskiyouensis in the field (left) were similar to symptoms observed in the inoculation trial (right). Symptoms in the field were more dramatic, in this instance, because they were on larger host trees and developed over a longer time...... …...…...... 143

4.6 Alder canker lengths in the pathogenicity test- Black circles represent the ratio of the medians of canker length adjoined to 95% CI error bars. Red hash line represents no difference from the negative control response. Mean canker length for the negative control was 0.1 cm. Red diamonds represent mean canker length after a 15 day incubation period. The gray area suggests an area of canker lengths resulting from a weak pathogen..……...... 145

LIST OF FIGURES (Continued)

Figure Page

4.7 The inner bark of bleeding cankers caused by P. siskiyouensis on alder looked very different from cankers associated with beetles or from Neonectria. From left to right – Phytophthora canker stacked with multiple dark boundaries (arrows), bore holes cankers from insects, and Neonectria canker with a faint boundary (arrow)...... 147

5.1. The 2010 alder health survey transects (red circles) in the three designated sub regions. The Willamette Valley (wv), southern (s) and coastal (c) (Chapter 2)...... 157

5.2 Red alder leaf debris (bottom) samples were collected in streams beneath tree canopies (top left) where red alder trees were the main source of leaf debris in streams. Leaf debris pieces were cultured on Phytophthora selective media (top right), plates were incubated and checked for colonies (arrow) growing from leaf pieces.………….…..…...... 167

5.3 Two transects (red arrows) were examined year round to confirm the apparent abundance of P. obrutafolium from streams and to examine a potential relationship with red alder stream debris. Transects were chosen where red alder trees were the main source of leaf debris in streams...... 168

5.4 Four isolates from a subcultured leaf debris sample. Each isolate was selected from a different red alder leaf in stream water in western Oregon. Isolates from a sample were selected for quantification based on their unique colony morphology. Each of the four colonies pictured above was considered unique...... 170

5.5 Average radial growth of P. obrutafolium after 96 hours of incubation at different temperatures. Error bars represent± 1 standard error...... 173

5.6 Mean radial growth of P. obrutafolium, P. gonapodyides and P. bilorbang on CA media following a 96 h incubation period...... 174

5.7 P. obrutafolium sp. nov. sporangia and hyphae. Swollen hyphal branch point (upper left), external proliferation of a sporangium (upper right). From left to right (bottom): internal extended proliferating growth, ovoid sporangium and, internal nested proliferation of a sporangium. Obpyriform sporangium with bulbous tip (inset). Bar = 50 u...... 177

LIST OF FIGURES (Continued)

Figure Page

5.8 The colony growth of P. obrutafolium sp. nov. (isolate 31-W-1.9 upper left) was much slower compared to other closely related species. P. taxon Pgchlamydo (isolate 2-50-R.5, upper right) P. lacustris (isolate 2-1-S.2, bottom left), and P. gonapodyides (isolate 2-W-1.20, bottom right)...... 178

5.9 Cox1 phylogeny showing the three closely related species...... 181

5.10 Map showing the locations of transects in western Oregon that were positive for P. obrutafolium...... 182

5.11 Isolates per liter of P. obrutafolium and other Phytophthora species from stream water for three season. Data is from two streams in western Oregon riparian ecosystems...... 184

5.12 Western Oregon recovery of Phytophthora. (a) - Phytophthora species recovered from streams associated with red alder. (b) - Phytophthora species recovered from red alder leaf debris...... 185

5.13 Red alder leaves artificially inoculated with P. obrutafolium zoospores. (Left) An empty sporangium indicating zoosporegenesis has occurred; sporangiophore and hyphae extend from the leaf surface. (Top right) Above the leaf surface several sporangia were observed. (Bottom right) A sporangiophore with sporangium was observed attached to the leaf surface...... 186

LIST OF TABLES

Table Page

2.1 Pathogens targeted for observations in the 2010 examination of alder trees in western Oregon riparian ecosystems.………………………………...... 25

2.2 Alder insects targeted in the 2010 examination of alder trees in western Oregon riparian ecosystems. Hopkins numbers are associated with Western Forest Insect Collection vouchered specimens in the Oregon State Arthropod Collection...... 27

2.3 Damage codes recorded for all alder trees ≥ 5 cm DBH in transects in western Oregon riparian ecosystems during the alder survey.…………………………...... 32

2.4 Summary data for alder trees in western Oregon riparian ecosystems. The n value is for the number of stands (transects). Note that dieback refers to canopy dieback. All values are averages...... 38

2.5 Proportion of transects containing alder trees with the observed damage indicators by sub-region and tree species in western Oregon riparian ecosystems…...... ……...39

2.6 2 x 2 contingency table values for damage indicator ‘yes’ values. Pearson’s chi square test, and Fisher’s exact test along with the odds ratio (θ), and the 95% confidence interval (CI)………………………………………………...... ……...….43

3.1 Phytophthora species recovered from different substrates in western Oregon riparian ecosystems. Three species were recovered from all substrates (gray rows)…………………………………………………………...... ……...... ……82

3.2 Substrate associations from western Oregon riparian ecosystems…...... 84

3.3 Base pair chart of Beta-tubulin double peaks……………………...... ……………....…..99

3.4 Phytophthora species recovered in western Oregon riparian ecosystems and reference matches…………………………………………………………...... …………….…..105

4.1 P. siskiyouensis, and P alni subsp. uniformis isolated in pure culture and recovered from diseased alder tissue. Isolates included were also recovered in association with alder stand with canopy dieback in western Oregon riparian ecosystems and include the two isolates of P. taxon Oaksoil used in pathogenicity testing...... 136

LIST OF TABLES (Continued)

Table Page

4.2 Comparisons of alder canopy dieback from transects with P. siskiyouensis vs. P. taxon Oaksoil…...... …...... ……139

4.3 Ratio (pathogen/control) of the medians estimate for canker length caused by the pathogen. The 95% confidence intervals (CI) based on estimates. Comparison is with n= 5 control treatments…………………………………...... ……....…140

5.1 Isolate information for Phytophthora species considered in this study...... 161

5.2 Cox 1 SNP chart comparing haplotype differences for three similar species...... 180

5.3 In western Oregon riparian ecosystems the number of transects in each sub-region positive or not for P. obrutafolium from baits and filtered stream water...... 183 1

Phytophthora Species and Riparian Alder Tree Damage in Western Oregon

Chapter 1. Dissertation introduction

In 2009, there were reports of alder trees (Alnus Miller (Betulaceae)) with

canopy dieback in riparian ecosystems in western Oregon. An unusual amount of alder

mortality was observed along the Smith River in Douglas County, Oregon. Alder trees

with bleeding cankers and canopy dieback were reported. Further observations in

western Oregon suggested that the mortality might not be confined to one area.

Historically, damage to alder in the western United States was caused by pathogens,

insects, and other wound agents such as ice (Worthington and Ruth 1962, Furniss and

Carolin 1977, Filip et al. 1989). Concerns about the health of alder trees were amplified in Oregon because of recent disease problems of alder in other parts of the world. In

Europe, in Australia, and other locations outside of western Oregon in the United States, pathologists were contending with diseases of alder trees caused by various organisms, including species of Phytophthora de Bary, Cytospora Ehrenb., and Melampsoridium

Kleb. In 1993, an invasive pathogenic Phytophthora species was found killing alder trees in Southern Britain (Gibbs 1995). A new species, Phytophthora alni Brasier & S. A. Kirk

(Brasier et al. 2004) was described, with three subspecies – subsp. alni, subsp. uniformis, and subsp. multiformis (Brasier et al. 2004, Ioos et al. 2006). Phytophthora alni subsp. 2 alni Brasier & S. A. Kirk was considered the most virulent subspecies (Brasier 2003), and by 2003 had affected or killed an estimated 15% of alder across southern Britain

(Webber et al. 2004). Phytophthora alni subsp. uniformis Brasier & S. A. Kirk was also found in riparian soils in Alaska in association with Alnus incana (L.) Moench (Adams et al. 2008) but was not associated with any dramatic disease there. There were no known

Phytophthora disease problems of alder in natural ecosystems in the United States at the time. However, a new Phytophthora disease had been found in planted Alnus cordata (Lois.) Duby, Italian alder trees in California. It was a concern that Phytophthora alni or another Phytophthora species could be causing disease in western Oregon, and that this disease would go undetected because riparian areas, where most alder occur, are not normally monitored for disease problems. This was especially disconcerting because red alder (Alnus rubra Bongard), the most common alder species in Oregon, is

Oregon’s most economically viable hardwood timber species, and provides irreplaceable ecological services in riparian ecosystems. This dissertation explores the diversity of

Phytophthora species in western Oregon riparian ecosystems, and their possible roles in alder canopy dieback.

THE GENUS PHYTOPHTHORA Phytophthora de Bary is a eukaryotic genus of plant pathogens unrelated to true

Fungi. Phytophthora species have some morphological similarities to Fungi. For example, Phytophthora species produce hyphae and spores. However, the genus 3

Phytophthora is genetically different from Fungi. Phytophthora is grouped with the

Stramenopiles in the super group SAR that also includes Alveolates and Rhizaria (Baldauf

2008). The genus Phytophthora is more closely related to brown algae than it is to true

Fungi. Anton de Bary first described Phytophthora as a genus in 1876 to separate it from

the genus Peronospora Corda and describe the potato late blight pathogen

Phytophthora infestans (Mont.) de Bary. Phytophthora is in the Peronosporales and

Peronosporaceae (Hulvey et al. 2010a, Thines 2013).

Phytophthora species cause many important agricultural plant (deBary 1876,

Hildebrand 1959, Werres et al. 2001, Hansen et al. 1979, Durán et al. 2008) and forest

tree diseases (Leonian 1925, Tucker and Milbrath 1942, Hansen et al. 1980, Gibbs et al.

1995, Rizzo et al. 2002, Goheen et al. 2002, Hansen et al. 2003, Jung et al. 2003, Brasier

et al. 2003b, Brasier et al. 2005, Greslebin et al. 2005, Brasier and Webber 2010, Brasier

2013). Phytophthora species rank prominently in lists of threats to forest tree health.

Examples include oak decline in Europe (Hansen and Delatour 1999, Jung et al. 2005),

and eastern North America (Hwang et al. 2008), sudden oak death caused by

Phytophthora ramorum Werres, De Cock & Man in’t Veld in the western United States

(Goheen et al. 2002, Rizzo et al. 2002), and alder decline caused by Phytophthora alni

Brasier & S. A. Kirk in Europe (Gibbs et al. 1995).

In forests especially, Phytophthora species may be present, even abundant, without causing significant disease. Work has been done on Phytophthora species in natural ecosystems (Hansen and Delatour 1999, Brasier et al. 2003a, Brasier et al.2003b, Hansen et al. 2009, Hwang et al. 2009, Remigi et al. 2009, Hulvey et al. 2010b, Reeser et al.

2011, Hansen et al. 2012a, Sims and Hansen In Press). Phytophthora species in Oregon forests in particular have been studied (Sutton et al. 2009, Reeser et al. 2011, Hansen et al. 2012b, Sims and Hansen In Press), but understanding Phytophthora species in ecosystems is complex, and which species and why they occur is often unknown

(Hansen 2012a).

Field diagnosis of plants infected with Phytophthora species can be difficult, and relies on examining disease symptoms, taking samples, sometimes sampling repeatedly, and culturing sampled material to attempt isolation. Field diagnostic tests can be expensive and impractical if hundreds or thousands of trees must be examined.

Phytophthora does not produce fruiting bodies which would provide evidence of presence, whereas many true Fungi, for example most of the basidiomycetes, do.

Characteristic symptoms of Phytophthora disease on hardwood trees include phloem island lesions (Brown and Brasier 2007) surrounded by a dark outer boundary. To successfully isolate the causal agent from the diseased host, rigorous sampling is often necessary, because of host phenolic compounds that inhibit the growth of Fungi 5

(Christie 1965) and Phytophthora species (Hüberli et al. 2000). If Phytophthora is

sampled and successfully grown in a laboratory setting, then the species can be

identified using morphological and molecular techniques. Further, an isolated individual

can be tested for its pathogenicity. A single organism, without the many extraneous

factors that occur in a natural setting, can be tested in a controlled environment to

determine if the observed field symptoms are produced under controlled conditions

using the standards of Koch’s postulates.

Three species of Phytophthora figure prominently in this dissertation:

Phytophthora alni Brasier & S. A. Kirk, Phytophthora siskiyouensis Reeser & EM Hansen

and Phytophthora taxon Oaksoil. The alder Phytophthora is Phytophthora alni. It causes

a lethal root and collar disease of alder species in Europe. It is especially devastating in

southern Britain (Gibbs et al. 1995) and it causes alder disease elsewhere in Europe. The

alder Phytophthora can be isolated from locations where it is causing disease by using baiting techniques, which are used to isolate it from soil, roots, and stream water. It can also be isolated from bleeding bole cankers. It has not been found causing alder disease outside of Europe, but concern that it might be introduced to Oregon triggered the surveys described here.

Phytophthora siskiyouensis was discovered in 2003 while monitoring areas damaged by the invasive Phytophthora ramorum, (Reeser et al. 2007). Phytophthora siskiyouensis is a minor pathogen of tanoak (Notholithocarpus densiflorus (Hook. & Arn.) Manos et al.), and Oregon myrtle (Umbellularia californica (Hook. & Arn.) Nutt.; Reeser et al. 2007).

This pathogen is new to science and little is known about its pathology. It was described as a pathogen of tanoak shortly before it was discovered causing cankers on planted black alder (Alnus glutinosa (L.) Gaertn.) in Melbourne, Australia (Smith et al. 2006), and killing planted Italian alder (Alnus cordata (Lois.) Duby) in California (Rooney-Latham et al. 2007, Rooney-Latham et al. 2009). Isolates recovered from Italian alder caused cankers in artificial inoculations of red alder, and white alder that occur in Oregon

(Rooney-Latham et al. 2009), evidence suggesting it could be involved in the dieback of alder.

Phytophthora taxon Oaksoil proved to be the most abundant species in our surveys, and for that reason, features prominently in this dissertation. This survey is the only report of the abundant presence of this organism in any ecosystem. An ecological role as an aquatic alder leaf saprotroph is proposed (Sims and Hansen In Press, Chapter

5) and Phytophthora taxon Oaksoil is included in the pathogenicity testing (Chapter 4).

ALDER Alder (Alnus Miller (Betulaceae)) are hardwood pioneer plant species. Alder are distributed across the Northern Hemisphere, and their range extends into the Andes of the Southern Hemisphere (Chen and Li 2004). In North America, alder species occur in temperate forests, and northern boreal locations. In western Oregon, alder often occurs with a patchy distribution along streams, and can occur as a major component of riparian ecosystems from high to low elevation. Alder habit is as a tree or a shrub and some alder species are both. Alder trees occur throughout western Oregon from the

Cascade Mountains to the Pacific Ocean. Three alder tree species are native to western

Oregon. Alnus rubra Bongard (red alder) is the most common hardwood in the Pacific

Northwest (Harrington 1990; Niemiec et al. 1995), and is a major component of riparian ecosystems in western Oregon (Barker et al. 2002). Red alder also occurs in upland locations in western Oregon on disturbed sites and on planted sites. Alnus rhombifolia

Nuttall (white alder or California alder) is a small to medium sized tree found in riparian ecosystems with fine-textured soils (Uchytil 1989). The third species of alder that reaches tree form in Oregon is Alnus incana (L.) Moench ssp. tenuifolia (Nutt.) Breitung

(thinleaf alder), found only in the southeastern portions of western Oregon and is more common as a shrub. A fourth native alder species occurs in western Oregon, Alnus viridis

(Chaix) DC. ssp. sinuata (Regel) Á. Löve & D. Löve (sitka alder) but its habit is that of a shrub.

In western Oregon riparian ecosystems, alder trees grow in both pure and mixed stands. In mixed stands, alder trees occur with other hardwoods such as Acer macrophyllum Pursh (big leaf maple), Populus balsamifera L. spp. trichocarpa (Torr. & A.

Gray ex Hook.) Brayshaw (black cottonwood), and Fraxinus latifolia Benth. (Oregon ash).

Usually, just upslope from riparian areas but sometimes within, softwoods such as

Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir), Thuja plicata Donn ex D. Don

(western redcedar), Tsuga heterophylla (Raf.) Sarg. (western hemlock), and in coastal areas, Picea sitchensis (Bong.) Carrière (sitka spruce) occur with alder trees (Franklin and

Dyrness 1973).

Alder root systems provide stream bank stability (Jensen et al. 1995), which leads to the formation of riparian ecosystems. Alder is a pioneer plant species that inhabits areas inhospitable to most other trees. This includes newly forming stream banks that are little more than sand and gravel bars (Fonda 1974; Hawk and Zobel

1974). In addition, alder trees that line the stream banks can regulate stream water temperatures by acting as a ‘hat’ that reduces the amount of heat entering a stream from solar radiation (Beschta 1997). Bank stability and cooler temperature streams are important factors in riparian ecosystems in western Oregon, and alder trees provide these ecosystem services.

In the Pacific Northwest, red alder is the most commercially important hardwood

(Harrington et al. 1994, American Hardwood Export Council 2009) and is the most common hardwood in the Pacific Northwest (Harrington 1990; Niemiec et al. 1995). Red alder is utilized for making doors, cabinets, furniture, joinery and mouldings. Douglas-fir is the most important timber species in the Pacific Northwest. However, red alder saw logs fetch a similar price as Douglas-fir (Deal and Harrington 2006). In addition, red alder can be grown on sites infested with conifer specific fungal pathogens such as Phellinus weirii (Murrill) Gilb., (Nelson et al. 1978) that causes the disease laminated root rot of

Douglas-fir, making red alder a positive alternative in some areas (Deal and Harrington

2006).

ALDER DIEBACK IN OREGON

Concern for the health of riparian alder increased in recent years. Observations by the Oregon Department of Forestry and the Forest Service indicated that some declining alder trees had symptoms similar to Phytophthora disease including bleeding bole lesions with a dark outer boundary, and canopy dieback. It was suspected that disease might not be confined to one area. A survey was designed to determine the extent of the dieback problem in riparian ecosystems in western Oregon, and catalog possible causal agents. In 2010, surveying, funded by the Forest Health Monitoring

Program of the USDA Forest Service, Pacific Northwest Region, began. Alder trees were 10 examined for bole symptoms known to occur in Europe and caused by the alder

Phytophthora, Phytophthora alni and other possible causal agents. A major goal of the survey was to characterize the species assemblage and determine the pathogenicity of

Phytophthora species isolated from streams, soil, and trees.

DISSERTATION OBJECTIVES

This dissertation reports and analyzes the results of the alder surveys, and evaluates the possible roles of Phytophthora species in the dieback observed. A goal was to gather baseline data on damage and damaging agents of alder trees. The first objective was to record and assess damage to alder trees in western Oregon riparian alder ecosystems. The second objective was to determine and evaluate Phytophthora species in western Oregon riparian alder ecosystems while monitoring for Phytophthora alni. The third objective was to evaluate damage caused by Phytophthora infection of alder in western Oregon. The fourth objective was to examine ecological associations of

Phytophthora to alder trees in western Oregon riparian ecosystems. To address the first objective damage was recorded and analyzed from 88 transects containing alder trees with canopy dieback symptoms. Trees were evaluated for damage caused by pathogens, insects, or wounds (Chapter 2). To address the second objective Phytophthora species from western Oregon riparian ecosystems were systematically sampled, isolated, identified, stored and compared (Chapter 3). To address the third objective Koch’s 11

Postulates were evaluated for three key Phytophthora species recovered and alder disease in the western United States was described (Chapter 4). To address the fourth objective the ecological role of the most abundant Phytophthora species from streams was evaluated and a new species is described (Chapter 5).

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Chapter 2. Phytophthora siskiyouensis and other damaging agents of alder trees in western Oregon riparian ecosystems

INTRODUCTION

In 2009, there were reports of alder trees (Alnus Miller (Betulaceae)) with

canopy dieback in riparian ecosystems in western Oregon (Figure 1). Historically,

damage to alder in the western United States was caused by pathogens, insects, and

other wound agents such as ice (Worthington and Ruth 1962; Furniss and Carolin 1977;

Filip et al. 1989). Concern about alder health arose in Oregon because of recent disease

problems in other parts of the world. In Europe, in Australia, and in the United States

pathologists were contending with diseases of alder trees caused by various organisms,

including species of Phytophthora de Bary, Cytospora Ehrenb., and Melampsoridium

Kleb. In 1993, an invasive pathogenic Phytophthora species was found killing alder trees

in England (Gibbs 1995). A new species, Phytophthora alni Brasier & S. A. Kirk (Brasier et

al. 2004) was described, with three subspecies – subsp. alni, subsp. uniformis, and

subsp. multiformis (Brasier et al. 2004, Ioos et al. 2006). P. alni subsp. alni was

considered the most virulent subspecies (Brasier et al. 2003), and by 2003 had affected

or killed an estimated 15% of alder across southern Britain (Webber et al. 2004). P. alni subsp. uniformis was also found in riparian soils in Alaska (Adams et al. 2008) but was

not associated with any dramatic disease. 20

Figure 1) In western Oregon riparian ecosystems red alder trees with canopy dieback.

Another Phytophthora species, P. siskiyouensis Reeser & EM Hansen, was

recovered from dying planted Alnus glutinosa (L.) Gaertn. (black alder) in Melbourne,

Australia (Smith et al. 2006) and from planted, dying Alnus cordata (Lois.) Duby (Italian

alder) in California (Rooney-Latham et al. 2009). P. siskiyouensis was first described from blighted Umbellularia californica (Hook. & Arn.) Nutt. (myrtlewood) shoots and from

Notholithocarpus densiflorus (Hook. & Arn.) Manos et al. (tanoak) bark cankers in

Oregon forests but was not seen on alder in the area, and no recovery from alder in

Oregon had been described (Reeser et al. 2007).

Valsa (cytospora) canker was reported as epidemic on thinleaf alder in the Rocky

Mountains in the United States (Worrall et al. 2010) and was implicated in thinleaf alder

dieback in Alaska and in the western United States (Stanosz et al. 2011).

Melampsoridium rust was reported as epidemic in parts of Europe on thinleaf alder

(Pôldmaa 1997; Hantula et al. 2009).

In the western United States, alder trees are ecologically critical in riparian

habitats and in the Pacific Northwest Alnus rubra Bongard is the most commercially

important hardwood (Harrington et al. 1994). Despite problems elsewhere, and the importance of the tree locally, no systematic pathology surveys had been done to provide baseline observation data on agents affecting alder trees. In 2010, such a survey was conducted in western Oregon riparian ecosystems to examine damage to alder 22 trees associated with pathogens, insects, and wounds. The Forest Health Monitoring

Program of the USDA Forest Service Pacific Northwest Region, the Oregon Department of Forestry, and Oregon State University cooperated on the survey. The survey focused on stands exhibiting canopy dieback and trees were examined for bole symptoms that have been associated with P. alni in Europe. Observations of pathogens, insects, and physical wounds on the boles and in the canopies of alder were recorded and statistical associations with canopy dieback were calculated.

Alder trees are hardwood pioneer plant species and distributed across the

Northern Hemisphere, extending into the Andes of the Southern Hemisphere (Chen and

Li 2004). In North America, alder occurs in temperate forests, and northern boreal locations. In western Oregon, alder trees often occur with a patchy distribution along streams, and can occur as a major component of riparian ecosystems from high to low elevation. Alder trees occur throughout western Oregon from the Cascade Mountains to the Pacific Ocean.

Three alder tree species are native to western Oregon. Alnus rubra Bongard (red alder) is the most common hardwood in the Pacific Northwest (Harrington 1990;

Niemiec et al. 1995), and is a major component of riparian ecosystems in western

Oregon (Barker et al. 2002). Red alder also occurs in upland locations in western Oregon on disturbed sites and on planted sites. Alnus rhombifolia Nuttall (white alder) is a small 23 to medium sized tree found in riparian ecosystems that shares many of the attributes of red alder (Uchytil 1989). The third species of alder that reaches tree form is Alnus incana

(L.) Moench ssp. tenuifolia (Nutt.) Breitung (thinleaf alder), found only in the southeastern portions of western Oregon. A fourth native alder species in western

Oregon is Alnus viridis (Chaix) DC. ssp. sinuata (Regel) Á. Löve & D. Löve, but, because it is a shrub, it was not included in this survey.

Gray ex Hook.) Brayshaw (black cottonwood), and Fraxinus latifolia Benth. (Oregon ash).

Usually, just upslope from riparian areas but sometimes within, softwoods such as

Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir), Thuja plicata Donn ex D. Don

(western redcedar), Tsuga heterophylla (Raf.) Sarg. (western hemlock), and in coastal areas, Picea sitchensis (Bong.) Carrière (sitka spruce) occur with alder trees (Franklin and

Dyrness 1973).

Riparian ecosystems are formed because successful pioneer tree species like alder inhabit areas inhospitable to most other trees. This includes newly forming stream banks that are little more than sand and gravel bars (Fonda 1974; Hawk and Zobel

1974). In riparian ecosystems, root systems of alder trees provide increased bank 24 stability (Jensen et al. 1995). In addition, alder lining stream banks can regulate stream water temperatures by acting as a ‘hat’ that reduces the amount of heat entering a stream from solar radiation (Beschta 1997). Bank stability and cooler temperature streams are important factors in riparian ecosystems in western Oregon, and alder trees provide these ecosystem services.

Pathogens

Numerous fungal pathogens and saprotrophic decay fungi occur on alder trees in western Oregon. The Pacific Northwest Fungi Database containing fungal host combinations lists 167 fungi with red alder, 16 fungi with white alder, and 102 fungi with thinleaf alder (Glawe 2009, Shaw 1958, http://pnwfungi.wsu.edu/programs/searchHostPerspective.asp). However, the list of pathogens targeted for observations in the survey (Table 1) was based on literature reports of damage to alder in western North America and is much shorter. Numerous insects have been recorded from alder trees in western forests. From the Western

Forest Insect Collection (USDA Forest Service, Oregon State Arthropod Collection

[OSAC]) the number of insect host combinations by alder tree species was: red alder 52, white alder 12, and thinleaf alder 9.

Table 1) Pathogens targeted for observations in the 2010 examination of alder trees in western Oregon riparian ecosystems. Name and Authority Damage Taxonomic/Damage Reference Canopy Chondrostereum purpureum Persoon 1794; Pouzar 1959 / silver leaf symptoms (Pers.) Pouzar Allen et al. 1996 Léveillé 1851; Hedwig 1805 / Erysiphe R. Hedw. ex DC. minor leaf damage Sims unpublished Klebahn 1899 / Poldmaa 2009; Melampsoridium Kleb. leaf browning, leaf damage Gjærum 2004 Dearness & Bartholomew Mycopappus alni (Dearn. & 1917; Redhead and White Barthol.) Redhead & G.P. large brown foliage spots 1985 / Redhead and White White 1985 Ellis and Everhart 1894; Septoria alnifolia Ellis & necrotic foliage spot Dearness / Constantinescu Everh. 1984; Harrington 2006 Taphrina occidentalis W.W. catkin deformity Ray 1939 / Mix 1949 Ray Kusano 1909, Ray 1940 / Mix Taphrina japonica Kusano deformed curling leaves 1949 Stem Armillaria sinapina Bérubé & Bérubé and Dessureault 1988; stem decay Dessur. Peck 1900 / Allen 1993 Ehrenberg 1818; Fries 1849 / Cytospora Ehrenb. stem and branch cankers Filip et al. 1992 Didymosphaeria oregonensis minor canker on young Goodding 1931 / Goodding Goodd. trees 1931 Wollenweber 1917; Neonectria major (Wollenw.) Perennial target shaped Wollenweber, H.W. 1926 / Castl. & Rossman cankers Cootsona 2006 Karsten 1881; Patouillard 1900 Phellinus ferruginosus white laminated rot of dead / Gilbertson and Ryvarden (Schrad.) Pat. wood 1987 white rot of heartwood of Fries 1849; Quelet 1886 / Phellinus igniarius (L.) Quél. living trees Gilbertson and Ryvarden 1987 Phytophthora alni ssp alni* Brasier et al. 2004 / Gibbs bleeding lesions / collar rot Brasier & S. A. Kirk 1995; Brasier 2003 Phytophthora alni ssp Brasier et al. 2004 / Gibbs multiformis Brasier & S. A. bleeding lesions / collar rot 1995; Brasier 2003 Kirk Phytophthora alni ssp Brasier et al. 2004 / Gibbs bleeding lesions / collar rot uniformis Brasier & S. A. Kirk 1995; Brasier 2003 Phytophthora siskiyouensis Reeser et al. 2007 / Rooney- bleeding lesions / collar rot Reeser et E. M. Hansen Latham et al. 2009, Sims 2013 Pleurotoid fungi white rot in dead wood / Brown and Gibson 2003 26

Insects

Many insects including beetles, moths, and sawflies are known to cause damage

to alder. Flea beetles cause periodic and severe damage to alder in the United States.

The flea beetle Altica ambiens LeConte is the most common insect defoliator of red alder in western Oregon (Filip et al. 1998). Alder bark beetles and ambrosia beetles can cause damage to healthy trees if large amounts of woody debris or slash accumulate

(Gara et al. 1978). Lepidopteran species of Malacosoma Hübner have caused periodic

high levels of defoliation in the past (Worthington and Ruth 1962; Gara et al. 1978). The

sawfly Monsoma pulveratum (Retzius) is the most recent of several alder sawfly

introductions to the United States; and it is considered an invasive defoliator of alder

(Smith and Goulet 2000; Kruse et al. 2010). Insects reported to cause damage to alder

species in western North America were summarized by Furniss and Carolin (1977), and

were the main target of observations in the survey; however we were also looking for

the invasive M. pulveratum (Table 2). Many of these insect species have vouchered

specimens from the Western Forest Insect Collection at OSAC (Table 2,

http://osac.science.oregonstate.edu/Sims_Alnus).

Table 2) Alder insects targeted in the 2010 examination of alder trees in western Oregon riparian alder ecosystems. Hopkins numbers are associated with Western Forest Insect Collection vouchered specimens in the Oregon State Arthropod Collection. Name and Authority Insect Type Damage References / Hopkins # Canopy Altica ambiens alder flea beetle / leaf skeletonizing and Furniss and Carolin 1977, LeConte leaf beetle leaf holes Filip et al. 1998

Eriocampa ovata alder woolly leaf skeletonizing of Furniss and Carolin 1977 (Linnaeus) sawfly lower leaves Furniss and Carolin 1977, Epinotia albangulana foliage feeder, leaf roller leafroller Miller and Hammond (Walsingham) and catkin damage 2002 / 13200 Hemichroa crocea foliage damage and Furniss and Carolin 1977 / the striped sawfly (Geoffroy) defoliation 31651 Furniss and Carolin 1977 / Malacosoma disstria forest tent defoliation Miller and Hammond / Hübner caterpillar 18842 Malacosoma Furniss and Carolin 1977 / californicum western tent defoliation Miller and Hammond / (Packard), syn. = M. caterpillar 31833 pluviale Monsoma green alder Smith and Goulet 2000, invasive defoliator pulveratum(Retzius) sawfly Kruse et al. 2010 Stem flathead wood Furniss and Carolin 1977, Agrilus burkei Fisher branch and twig damage borer Harrington 1990 / 4759 Alniphagus aspericollis mine inner bark of Furniss and Carolin 1977 / alder bark beetle (LeConte) damaged trees 18179 Worthington et al. 1962, Gnathotrichus alni bore holes in main stem ambrosia beetle Furniss and Carolin 1977 / Blackman of weak or downed trees 18179 Trypodendron Worthington et al. 1962, lineatum (Olivier), syn. bore holes in main stem ambrosia beetle Furniss and Carolin 1977 / inferred = T. cavifrons of weak or downed trees 18861 (Mannerheim) mine the inner bark of Furniss and Carolin 1977 / Trypophloeus species bark beetle living tree boles and 16324 large branches Xyloborus arbuti Worthington et al. 1962, bore holes in main stem Hopkins, syn. = X. ambrosia beetle Furniss and Carolin 1977 / of weak or downed trees saxeseni (Ratzeburg) 31697

Wounds

Alder stands in Oregon have been damaged by ice storms, as indicated by broken

tops (Worthington and Ruth 1962). In eastern Oregon and Washington, floating ice

sheets in streams may cause severe wounds to trees in riparian areas (Filip et al. 1989),

but this type of damage is unlikely to occur in western Oregon where winter temperatures are much warmer. Other objects propelled during flood events could cause wounds to trees in riparian areas. Nearby falling trees can also damage an alder tree. Other potential damaging agents are humans, bears, elk, and deer; and in riparian

areas, beavers can act as wounding agents. Fire is not considered important in damaging

alder. In fact, a management strategy is to plant alder as a fire break (Harrington 1990).

It has been suggested that wounded alder trees (Allen 1993, Filip et al. 1989) are more

likely to be colonized by decay fungi because wounds provide entry courts for decay

causing organisms.

The objective of the survey was to provide baseline knowledge on damage from

pathogens, insects and wounds associated with alder canopy dieback in western Oregon

riparian ecosystems.

MATERIALS AND METHODS

Transects

Riparian alder stands in western Oregon (west side of the Cascade Mountains to the Pacific Ocean) were surveyed between 2 June 2010 and 19 October 2010 along 88,

100 m long by 10 m wide transects. The area was divided into three sub-regions: the

Willamette Valley sub-region with streams draining into the Willamette River (33 transects); the southern sub-region in SW Oregon (25 transects), and the coastal sub region with streams draining into the Pacific Ocean (30 transects) (Figure 2). The area was divided to assure reasonably uniform sampling across all of western Oregon.

Transects were not closer than 0.5 km linear distance from any other transect. Transects were selected to include alder trees exhibiting dieback. Other selection criteria included access and bank stability. 30

Figure 2) Locations of transects in western Oregon riparian ecosystems for the alder survey. Latitude and longitude of the southernmost transect (224): N° 42.0060, W° 124.2132. 31

A subset of 18 of the established transects were revisited in December 2011

through March 2012 and in October 2012, to follow up on particularly interesting

symptoms, especially bleeding cankers, observed in the main survey. There was an

attempt to resample the same trees from the original survey; however, this was only

done if the same trees could be easily relocated and if they were the trees with the best

canker symptoms. Tissue pieces from symptomatic samples were plated on selective

media for recovery of Oomycetes, particularly Phytophthora species CARP (cornmeal agar, 10 ppm rifamycin SV sodium salt, 20 ppm Delvocid [50% natamycin salt], 200 ppm ampicillin sodium salt) (Reeser et al. 2011). Five alder trees from each transect were sampled.

Data Collection

Information was recorded for alder trees with a > 5 cm DBH including: tree species, general condition, DBH in 5 cm classes, percent canopy dieback in 10% increments for living canopy dominants, visible indicators of damage (not minor occurrences, Table 3), and remarks. Canopy dieback (Figure 1) was due to dead or dying branches and branchlets affecting areas of the canopy that were not shaded (top of the canopy downward or exposed to accessible light). Trees with chlorotic or sparse foliage were noted. Insect or leaf disease defoliation was not considered canopy dieback.

Understory alder were not scored for canopy dieback since alder trees are shade 32 intolerant. However, understory alder trees were still tallied and DBH was recorded.

Trees were scored as having dieback or not and dieback was defined as the proportion of live canopy missing due to dead or dying branches. Alder tree condition was recorded as living, recently dead (with small attached branches), or old dead. Trees were tagged near the base of the bole with transect and tree number.

Table 3) Damage codes recorded for all alder trees ≥ 5 cm DBH in transects in western Oregon riparian ecosystems during the alder survey. Location on Symptom or Damage Agents Tree Code Damage Type Basal canker or Bleeding Lesion Pathogens stem SC1 (Phytophthora canker) Ascomycete Stem SC2 canker SD Stem decay canopy FPR Foliar disease: rust Foliar disease: FPS leafspots FPO Foliar disease: other Insects stem SI Stem boring canopy FIC Foliage: chewing FIO Foliage: other Wounds stem SW Stem wound canopy BT Broken top

Damaged plant material and/or insects were collected as necessary for identification. Damage was recorded when it affected a significant portion of the alder tree, either in the canopy or in the stem. Only visible symptoms of obvious damage 33

were recorded. Identification of pathogens and insects was by morphology and damage

patterns (Tables 1 and 2). The group ‘other’ for insect and pathogen related damage

was included to avoid potential oversight.

Bark samples were collected from trees with cankers and plated on Oomycete

selective media (CARP). For Phytophthora positive cultures a clean hyphal branch was sub-cultured to corn meal agar with 20 ppm β-sitosterol (CMAβ) (Reeser et al. 2011).

Morphological characterization of Phytophthora species was done on culture plates

(CARP and CMAβ). Phytophthora positive cultures were placed in water storage. For

species level identification, DNA was extracted, PCR amplified and sequenced in the

cytochrome c oxidase (Cox) spacer region (Martin and Tooley 2003) of the mitochondrial

DNA. Sequences were manually examined and aligned against an in-house library of

Phytophthora sequences.

Data Analysis

The data were used to compare the associations between canopy dieback and

damaging agents of alder trees. We examined the odds ratios that alder tree canopy

dieback was associated with (1) pathogen indicators, (2) insect indicators, and / or (3)

wound indicators, as well as (4) the odds ratio that stem wounding was associated with

stem decay, and (5) whether there were similarities in damage observed for alder trees

across western Oregon. 34

In order to address the first three damage factors, 2 x 2 contingency tables

(Agresti 2007) were constructed for dieback and damage. Damage was grouped based

on the symptom or type and location of the damage on a tree. To examine the

association between wounding and stem decay, wounding was compiled into 2 x 2

contingency table format with stem decay. Analysis included data from all living alder

trees in the survey.

The sample odds ratio was defined as:

π1 (1−π ) θ = 1 π 2 (1−π 2 )

Where success (π) was the proportion of trees with canopy dieback or stem

decay. The probability of success with damage was π1, and the probability of success

without damage was π2. It was assumed that total sample sizes were large enough, and

that each value fit exactly into only one cell.

In R version 2.15.2 (R Core Team 2012) 2 x 2 matrices were constructed with the

table values. Then Fisher's exact test for contingency tables (Fay 2010) and the Pearson's

Chi-squared test (Agresti 2007) were performed for each matrix. Summary data for alder 35 trees in stands were generated for tree species by sub-region (Table 4). Summary data were also compiled (Table 5) to examine similarities in damage factors across western

Oregon. Table 6 contains the data for odds ratio analysis.

RESULTS

Of 2308 living alder trees observed 1,445 (62%) were red alder, 682 (30%) were white alder, and 181 (8%) were thinleaf alder. Red alder occurred in stands from all three sub-regions, and was the only alder species in stands in the coast sub-region.

White alder occurred in stands in the southern sub-region and in the Willamette Valley sub-region. Thinleaf alder occurred in stands in southern sub-region only. There were

421 dead alder trees observed in total, of which 92 had recently died.

Canopy Dieback

Canopy dieback was generally recent (Figure 3). The dieback was considered recent because the trees maintained dead branches and branchlets. Trees with old broken tops often had full canopies with little to no apparent dieback. Of the living trees, 42% had canopy dieback symptoms, but this varied by sub-region and alder species. Alder canopy dieback was the greatest overall (66%) on red alder in the coast sub-region (Table 4). For white alder dieback was the greatest (33%) in the Willamette

Valley sub-region, but did not substantially differ from white alder dieback in the southern sub-region (24%, Table 4). For most species and sub-regions, trees with dieback were no larger than apparently healthy trees (Sims and Hansen 2012). In the

Willamette Valley sub-region, however, red alder trees with dieback symptoms were 37 somewhat larger on average than trees without dieback (Table 4); this was mainly due to trees with ≤10 cm DBH without dieback.

Figure 3) A red alder tree with recent canopy dieback in western Oregon riparian ecosystems. Note: a standing dead alder tree is pictured left of the tree with canopy dieback.

Table 4) Summary data for alder trees in western Oregon riparian ecosystems. The n value is for the number of stands (transects). Note that dieback refers to canopy dieback. All values are averages.

Number Proportion of trees DBH of DBH of of trees per trees with trees with with Tree Species Sub-region transect dieback no dieback dieback n thinleaf alder southern 60 10 10 0.10 3 white alder southern 52 15 20 0.24 12 white alder valley 21 30 30 0.33 10 red alder coastal 19 35 30 0.66 30 red alder southern 41 20 20 0.38 11 red alder valley 15 30 20 0.46 29

Pathogens

Canopy - Foliar leafspot disease in the coast sub-region occurred in nearly all stands (97%, Table 5) and in about 45% of alder trees, but was less frequent in stands in the other two sub-regions and occurred on only 5% of trees. Foliar leaf spot symptoms were examined more closely on trees from the coast sub-region. Two ascomycetes were identified: Septoria alnifolia Ellis & Everh. (Figure 4), and Mycopappus alni (Dearn. &

Barthol.) Redhead & G.P. White (Figure 4). S. alnifolia leaf spots were pale brown and circular to irregular, generally 1-2 cm in diameter with a darker margin, and contained clusters of pycnidia in the centers of the leaf spots. M. alni conidiomata were white, mop - like, and consisted of filamentous tufts of conidia that were connected to the leaf tissue. White specks (observed with the naked eye) over the brown leaf blotch symptom were M. alni conidiomata. Leaf spots ranged in size from 1-5 cm in diameter and were very irregular in shape. Specimens were deposited in the Oregon State University

Herbarium (OSC). 39

Table 5. Percentage of transects containing alder trees with the observed damage indicators by sub region and tree species in western Oregon riparian ecosystems.

Damage Indicators: Pathogens Insects Wounds foliage stem foliage stem top stem stands Tree Species sub-region FPS FPR FPO SD SC1 SC2 FIC FIO SI BT SW n Alnus incana * southern 33% 0% 0% 0% 33% 100% 67% 67% 33% 100% 100% 3 Alnus rhombifolia southern 25% 0% 17% 58% 42% 33% 33% 8% 33% 33% 100% 12 Alnus rhombifolia W. Valley 50% 0% 20% 40% 30% 50% 0% 20% 30% 20% 50% 10 Alnus rubra coast 97% 0% 0% 43% 30% 27% 77% 13% 30% 40% 83% 30 Alnus rubra southern 27% 0% 18% 45% 45% 27% 82% 0% 36% 20% 100% 11 Alnus rubra W. Valley 34% 3% 3% 24% 10% 45% 72% 3% 24% 36% 59% 29

Foliage pathogens: FPS = spot, FPR = rust, FPO = other. Stem pathogens: SD = stem decay, SC1 = Phytophthora canker, SC2 = Ascomycete canker. Foliage Insect pests: FIC = chewing foliage insects, FIO = other foliage insects. Stem Insects: SI = stem or branch boring insects. Wounds: BT = broken top, SW = stem wound. For values: ≥ 25% cells are light grey, ≥ 50% cells are dark grey, and ≥ 95% cells are black. 40

Figure 4) Common foliar damage of alder in western Oregon (Left to right from top) - Ascomycete leaf spot pathogen Septoria alnifolia was recovered from alder trees with diseased leaves examined during the survey, clusters of pycnidia, were in the center of leaf spots. - Brown leaf blotch on a red alder leaf collected during the survey, note the white conidiomata of the ascomycete leaf spot pathogen Mycopappus alni. - Brown leaf blotch whole leaf symptom from a red alder trees with diseased leaves, the white spots are signs of the pathogen. - The most commonly observed insect damage of alder trees was from insects like the alder flea beetle Altica ambiens (larval stage). - Sawflies larva were common on alder leaves, the woolly alder sawfly larvae (Eriocampa ovata) feeding on an alder leaf. - Alder leaf damaged from feeding by sawfly larvae. White bar = 5cm. Black bar = 1 cm. 41

Foliar rusts were only counted as damage once, in the Willamette Valley sub

region, on a single red alder found along the McKenzie River. The group ‘other’ for foliar

pathogen related damage included leaf tip blight and leaf curl similar to that caused by

Taphrina japonica Kusano. Foliar pathogens ‘other’ were encountered infrequently (in <

25% of stands, Table 5). Catkin deformity with tongue like enlargements of the bracts of

female catkins was observed from nine white alder trees and was morphologically

identified as Taphrina occidentalis W.W. Ray some of the same trees had galled and

curled areas on the leaves. These symptoms were observed in southern Oregon and at a

location between two surveyed stands along a stream in the Willamette Valley. In a

return visit to a single stand in the late fall, not long before natural leaf senescence,

rusts and powdery mildews were common on leaves.

Stem - Bleeding lesions and basal cankers characteristic of Phytophthora species

bole infections (SC1 bleeding spots over necrotic phloem, typical of Phytophthora

symptoms) (Figure 5) occurred in more than a quarter of stands across western Oregon

(Table 5) and on 2.5% total trees (except in the Willamette Valley for red alder where

only 10% of stands had SC1). SC1 were observed on 57 alder trees (33 red alder, 23

white alder, and 1 thinleaf alder). The odds of an alder having both canopy dieback and

SC1 were 5.3 times greater than the odds of having canopy dieback without canker damage (p < 0.0001, Table 6). In the original survey, which occurred mainly during the summer months, only one isolation was successful, from the southern Oregon sub 42

region, from a site surveyed in October. P. siskiyouensis was isolated from a red alder

tree with 70% canopy dieback. The tree also had an ax wound but the damage was

considered minor, and the bleeding lesion did not appear to originate in the ax wound.

A follow-up survey focused on bleeding cankers similar to those observed in the main summer survey. In the follow-up survey, conducted mostly from December 2011 to

March 2012 in selected stands in the coast sub-region but also from one stand in the

Willamette Valley in October 2012 P. siskiyouensis was isolated 15 times from bark cankers. Recovery was from all three sub-regions from red alder and white alder. No other Phytophthora species were recovered from bole cankers. Bleeding cankers were

both small bleeding lesions (Figure 5, upper right) and large bleedings cankers extending

up the main bole of the tree with small bleeding lesions above the main larger canker

(Figure 5 arrows, left). Bleeding cankers were sometimes accompanied by dry, cracked

bark (Figure 5, bottom right).

Table 6) - 2 x 2 contingency table values for damage indicator ‘yes’ values. Pearson’s chi square test and Fisher’s exact test were computed for each relationship along with the odds ratio (θ), and the 95% confidence interval (CI). Canopy Damage Indicator Dieback Full Canopy Pearson / Fisher θ CI Pathogens: Phytophthora canker 45 12 <0.0001 / <0.0001 5.3 2.7, 11 Ascomycete canker 34 34 0.25 / 0.21 1.4 0.81, 2.3 stem decay 61 59 0.07 / 0.07 1.4 0.97, 2.1 Wounding: broken top 47 53 0.35 / 0.41 1.2 0.79, 1.8 stem wound 232 274 0.09 / 0.09 1.2 0.97, 1.5 Total trees: 981 1327 Stem No Stem Damage Indicator Decay Decay Pearson/Fisher θ CI Wounding: broken top 11 89 0.01 / 0.02 2.4 1.1, 4.6 stem wound 59 447 <0.0001 / <0.0001 3.8 2.5, 5.6 Total trees: 120 2188 44

Figure 5) Phytophthora cankers on alder trees in western Oregon riparian ecosystems. (Left) Alder tree with Phytophthora type canker (SC1) in an alder stand. Phytophthora siskiyouensis was recovered from the canker. The large bleeding canker extended up the bole well over 1.5 m in length with small bleeding cankers continuing to at least 2.5 m (arrows). (Top right) Alder tree with SC1 in an alder stand (chisel is for scale the blade is 3.7 cm wide). (Bottom right) SC1 ooze on the outside of the bark discolors the outer bark orange, red and dark grey; cankers were sometimes accompanied by dry cracked bark.

Stem cankers (SC2) like those typically caused by various ascomycete fungi

occurred widely across western Oregon (Table 5) and on 2.8% total trees. The odds of

an alder tree having both canopy dieback and SC2 damage were 1.4 times greater than

the odds an alder tree had canopy dieback without canker damage; this value was not

statistically significant ( p = 0.21, Table 6). Perennial target shaped cankers and early symptoms of cankers similar to those described by Cootsona (2006) for Neonectria

major (Wollenw.) Castl. & Rossman, were noted most frequently in the Willamette

Valley sub-region. Symptoms similar to rough bark canker (Goodding 1930) caused by

Didymosphaeria oregonensis Goodd. were observed on red alder from two transects,

one in the Willamette Valley sub-region, and the other in the southern Oregon sub

region. Cankers of this type were only observed on the smallest size class (2-4 inch DBH)

trees. Neither perennial cankers, nor rough bark cankers were noted frequently, nor did

they appear to be causing any major damage.

Basidiocarps of wood decay fungi on living alder trees were infrequently

encountered. Phellinus igniarius (L.) Quél was not identified. Phellinus ferruginosus

(Schrad.) Pat. was noted on attached dead lower branches and on fallen branches of

white alder. Attached dead branches with resupinate conks were not considered stem

decay damage, as the infection only appeared in branches. P. ferruginosus was also

observed on one large diameter living red alder with compartmentalized decay, it was 46

growing on the dead area of the tree, consistent with its understood role as a

saprotroph of dead wood of hardwoods (Gilbertson and Ryvarden 1987).

Pleurotoid fruiting bodies were observed on old dead trees, and were rarely on

large living trees with extensive decay. Observation of fruiting structures was limited by

the time of year when the survey was conducted. Stem decay was often noted in trees

with no obvious fungal signs, but was understood to be present due to woodpecker

cavities and / or the presence of obvious dead and decayed wood areas. Decayed areas

were easily recognized because they gave way when struck lightly with the blunt side of

a hatchet.

The odds of an alder having both canopy dieback and stem decay damage was

1.4 times greater than the odds of having canopy dieback without stem decay damage.

This was not a significant difference (p = 0.07, Table 6).

Insects

Canopy - The most common insect damage on red alder and thinleaf alder was associated with foliage feeding insects (> 50% of stands, Table 5) and was observed in

23% of alder trees. Of these, the most commonly observed were flea beetle larvae

(Figure 4) and sawfly larvae (Figure 4). Tent caterpillars (Malacosoma species) were infrequently observed on both red alder and white alder, but have been important 47 damage agents in the past (Worthington and Ruth 1962). Tent caterpillars were not observed on thinleaf alder.

All other foliage (FIO) insects affected < 2% of trees in any sub-region. Aphids were noted but not considered to be causing important damage. The group ‘other’ for insect related damage included leaf rolling insects, spittlebugs, and leaf gall insects. FIO damage occurred in 10 stands and on 33 trees.

Stem– Damage by stem insects such as ambrosia and bark beetles to alder trees was infrequent but widespread across western Oregon. Damage was recorded on 3% of alder trees, but occurred in a (transect weighted) mean average of 32% of stands.

Wounds

Canopy – Alder stands with broken tops were scattered across western Oregon, and broken tops occurred in 4% of alder trees. The average percentage of alder stands containing trees with broken tops ranged from 20% to 100%, and varied depending on the tree species and the sub-region (Table5). The odds that an alder had both canopy dieback and broken top damage were 1.2 times greater than the odds of having canopy dieback without broken top damage. Canopy dieback was not explained by broken top wounds (p = 0.4, Table 6). Most broken tops were the result of past ice storms. Many trees with old broken tops had regenerated full canopies. 48

Stem– Alder trees with stem wounds occurred frequently and stem wounds were widespread in stands. The average percentage of stands containing alder trees

with stem wounds was ≥ 50% (Table 5) in all cases. Damage from stem wounds was

recorded on 22% of alder trees. The odds that an alder tree had both canopy dieback

and stem wounds were 1.2 times greater than the odds of having canopy dieback

without stem wounds. Canopy dieback was not explained by stem wounds (p = 0.09,

Table 6).

Sapsucker damage was not frequent, but did occur heavily in a few stands in the

southern Oregon sub-region. Bear and beaver damage was observed infrequently.

Human damage was not common in these stands, but included chopping, damage from

wrapped wires, bark carving, and anthropogenic debris propelled into trees by floods.

Wounds and stem decay - There was a strong association between stem decay

and wounding. The odds of stem decay were 2.4 (broken top), and 3.8 (stem wound)

times greater in wounded trees compared to non-wounded trees (Table 6). Stem decay

was observed on about 5% of alder trees.

DISCUSSION

Mature alder trees with canopy dieback were commonly observed in western

Oregon. Although presence of dieback was a stand selection criterion in this survey, it

was not difficult to locate suitable transects. Alder health is of special concern because

of the important ecosystem roles of these trees, and because of reports of the

destructive epidemic of the invasive alder Phytophthora in Europe and recent reports of

a variant of P. alni in North America. Indeed, fear that the alder Phytophthora was

already in Oregon provided the motivating force for this project. Because P. alni is most readily identified in Europe from bleeding lesions on tree boles and subsequent dieback and tree death, our survey concentrated on similar bole symptoms. We then expanded the objectives to include an extensive assessment of damaging agents on alder trees and their association with canopy dieback. A parallel survey of alder root health was undertaken; those results will be reported separately.

Many of the pathogens targeted based on literature reports of damage to alder

(Table 1) were identified in the survey. Most notable by its absence from above ground parts of alder trees in western Oregon riparian ecosystems was P. alni subsp. alni.

Evidently, this destructive pathogen has not (yet) been introduced here. P. alni subsp. uniformis is known from Alaska (Adams et al. 2008), and was recovered occasionally from lesions on alder roots in the parallel soil-, root- and water- based survey (Sims in 50

prep, Aguayo et al. 2013). This subspecies is less aggressive to alder, at least in Europe

than P. alni subsp. alni (Brasier 2003), and may be native to western North America

(Aguayo et al. 2013).

Canopy Dieback

Dieback of riparian alder was widespread in western Oregon, but no single agent identified in this survey provided a likely explanation for all the canopy dieback. Based on odds ratio, Phytophthora canker was the most strongly associated with trees exhibiting canopy dieback. Affected trees were found in all three sub-regions and P.

siskiyouensis was isolated from the two main alder tree species in the survey (red alder

and white alder).

Pathogens

Canopy- Foliar pathogens have not been observed to cause important damage to

mature alder trees in natural settings, although Mycopappus alni in British Columbia has

been reported to be the cause of early defoliation in understory alder trees (Redhead

and White 1985), and Septoria alnifolia has been noted to cause economic losses in

nursery settings (Harrington 2006). In the coast sub-region of western Oregon foliar leaf

spot pathogens (Figure 4) may in fact be important pathogens, as evidenced by their widespread occurrence on a high proportion of trees.

Leaf rusts were not causing damage during the main part of the growing season when the survey was conducted, but in a return visit to a single stand in the late fall, not long before natural leaf senescence, rusts and powdery mildew were common on leaves. What effect late season damage of leaves has on tree health is unclear.

Stem - Phytophthora canker damage (Figure 5) had the strongest odds association with canopy dieback of any of the symptoms examined and SC1 damage was widespread but relatively infrequent. Isolation success may have been low in the main survey due to summer sampling. Additional recovery of P. siskiyouensis was successful in the follow up survey later in the year. With the subsequent return to 18 stands in the winter and spring, overall recovery of P. siskiyouensis from alder tree cankers was from all three sub-regions and from two alder tree species red alder and white alder.

Sometimes the bleeding cankers appeared to drain and ooze down the outside of the bark and severe bleeding discolored the bark (Figure 5). This is the first report of

Phytophthora canker on alder in natural stands in the Americas.

Ascomycete canker damage SC2 was not strongly associated with canopy dieback and occurred on few trees but damage was widespread. Cankers were not all thought to be caused by Neonectria or Didymosphaeria and many different ascomycete fungi might be involved in these types of cankers including Cytospora and Melanconis species. The association of ascomycete fungi may also be different depending on which 52

tree species are considered. Ascomycete pathogens in other locations in the western

United State are important in causing disease on thinleaf alder(Worrall et al. 2010;

Stanosz et al. 2011) including northeastern Oregon where thinleaf alder is much more common (Filip et al. 1992). In western Oregon, thinleaf alder is a minor component of the alder tree species that occur, and occurs mainly in the more mountainous part of the southeastern corner of western Oregon. The three thinleaf alder stands (and the

197 trees) examined had 1 or more thinleaf alder trees with SC2, and cankers did appear to be associated with canopy dieback. However only 9 thinleaf alder trees in total had stem cankers.

Decay damage was not statistically associated with canopy dieback and was infrequently observed. The fact that decay was not important in association with canopy dieback is probably, at least in part, because wound decay can be compartmentalized in living trees. P. igniarius, although reported as the most important decay fungus in alder, was not observed. Internal decay was not systematically sampled, however, and the lack of conks may not be a good indication of the amount of decay (Allen 1993).

Insects

Canopy - Foliar insects were damaging about a quarter of the alder trees surveyed. The defoliating insects observed here have caused periodic defoliation to 53 alder trees elsewhere. Damage was mainly attributed to Altica ambiens (Figure 4) and the larval stage of sawflies (Figure 4) such as Hemichroa crocerea and Eriocampa ovata.

Stem - Stem insects (Bole insects: bark beetles and wood borers) did not appear to be affecting many trees although when they were present it was mainly on larger

(older) alder trees with canopy dieback, consistent with the secondary nature of many bark and wood attacking insects. In a few locations, with heavy alder debris (log) accumulation there was heavy wood borer damage.

Wounds

Canopy - Broken tops were mainly attributed to ice storms. It has been suggested before that red alder is more susceptible to ice damage than conifers, but that trees appear to recover (Worthington and Ruth 1962) by regenerating a new canopy.

Stem - Most stem wounds apparently resulted from debris swept by the flow of high water into tree boles, because wounds were on the upstream side of trees. Other stem damage resulted from nearby falling trees. Wound damage usually did not appear to be severe enough to contribute to canopy dieback, but did appear to provide an entry pathway for stem decay fungi.

Wounds and Stem Decay - Wounding was an important indicator of stem decay for red alder. In another study on Vancouver Island in British Columbia, red alder scars were a good indicator of decay (Allen 1993). The results from the western Oregon survey support the conclusions that wounding (which would result in scars) is an important factor in relation to stem decay for alder trees in Oregon as well. Although there was no evidence that thinleaf alder spp. tenuifolia trees were affected by stem decay, the trees were very small suggesting the three stands examined were young stands. It could be that not enough time had elapsed since the damage occurred for decay to be evident.

Other

Another factor that was evaluated in this survey was drought or unusually low stream flow (Data not shown). Trees growing in normally moist soils such as riparian zones are particularly vulnerable to occasional droughts (Wolken et al. 2011). If this were the case then it was assumed that trees furthest away from the stream would be the most affected while trees closer to the water source would be less affected (Worrall

2009). The reverse was actually true and trees closer to the water source had more canopy dieback. A similar inverse relationship was found for dieback and mortality of

Alnus in the Southern Rocky Mountains (Worrall 2009). Possible effects due to high temperatures that may be independent of drought events could be important (Worrall

2010) and these effects were not examined. Alder is known as a short-lived tree 55

(Niemiec et al. 1995), but trees of all sizes (and presumably ages) exhibited dieback.

From this survey, it appears that the prevalence of dieback in riparian alder stands is the cumulative effect of individual trees affected by different agents.

CONCLUSIONS

• Phytophthora siskiyouensis was isolated from bole cankers of alder trees (red

alder and white alder) in western Oregon from all three sub-regions.

Phytophthora cankers were associated more than other damaging agents with

canopy dieback, and incidence in alder stands containing alder trees with canopy

dieback was 2.5%. Season of year apparently affects isolation success. This is the

first report of Phytophthora canker on alder in natural stands in the Americas.

• Phytophthora alni subsp. alni was not identified in western Oregon at this time.

• Many of the same damaging agents that have been recognized in the past on

alder trees in the western United States are also damaging to alder trees in

western Oregon riparian ecosystems.

• Foliage pathogens, only previously recognized as being important in a nursery

setting or in the canopy understory, may be important damaging agents in

western Oregon riparian ecosystems, at least in the coastal areas to red alder in

particular.

• Foliage insects, including many introduced species were causing damage to alder

trees, but this is not new and has been suggested by others to be cyclic.

• Wounding to alder trees in western Oregon riparian ecosystems was not an

important damaging factor in relation to canopy dieback but was important in

relation to stem decay. 57

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Chapter 3. Phytophthora species from riparian alder ecosystems in western Oregon, USA

INTRODUCTION

In western Oregon, red alder (Alnus rubra Bong., Betulaceae) is the most

common hardwood tree. It is ecologically important, and an economically viable timber

species. In 2009, an unusual amount of alder mortality was observed along the Smith

River in Douglas County, Oregon. Alder trees with Phytophthora cankers and canopy

dieback were reported. Further observations in western Oregon suggested that the

mortality might not be confined to one area. In Europe (Gibbs et al. 1999, Streito et al.

2002, Webber et al. 2004), in Australia (Smith et al. 2006), and in the United States

(Rooney-Latham et al. 2007) alarming new diseases of alder caused by species of

Phytophthora de Bary had recently been reported. The present work was undertaken to

provide a baseline of information on Phytophthora species presence and abundance in

streams, soils, and roots in alder riparian ecosystems in western Oregon.

Phytophthora is a eukaryotic genus of plant pathogens unrelated to true fungi. It

is grouped with the Stramenopiles in the super group SAR that also includes Alveolates

and Rhizaria (Baldauf 2008). Anton de Bary first described Phytophthora as a genus in

1876 to separate it from the genus Peronospora Corda and describe the potato late

blight pathogen Phytophthora infestans (Mont.) de Bary. Phytophthora contains many 64

important agricultural plant (deBary 1876, Hildebrand 1959, Werres et al. 2001, Hansen

et al. 1979, Durán et al. 2008) and forest tree pathogens (Leonian 1925, Tucker and

Milbrath 1942, Hansen et al. 1980, Gibbs et al. 1995, Rizzo et al. 2002, Goheen et al.

2002, Hansen et al. 2003, Jung et al. 2003, Brasier et al. 2003, Brasier et al. 2005,

Greslebin et al. 2005, Brasier and Webber 2010, Brasier 2013).

Phytophthora species rank prominently in lists of threats to forest tree health.

Examples include oak decline in Europe (Hansen and Delatour 1999, Jung et al. 2005)

and eastern North America (Hwang et al. 2008), sudden oak death caused by

Phytophthora ramorum Werres, De Cock & Man in’t Veld in the western United States

(Goheen et al. 2002, Rizzo et al. 2002), and alder decline caused by Phytophthora alni

Brasier & S.A. Kirk in Europe (Gibbs et al. 1995).

In forests especially, Phytophthora species may be present, even abundant,

without causing significant disease. Work has been done on Phytophthora species in natural ecosystems (Hansen and Delatour 1999, Brasier et al. 2003a, Brasier et al.2003b,

Hansen et al. 2009, Hwang et al. 2009, Remigi et al. 2009, Hulvey et al. 2010, Reeser et

al. 2011, Hansen et al. 2012a, Sims and Hansen In Press). Phytophthora species in

Oregon forests in particular have been studied (Sutton et al. 2009, Reeser et al. 2011,

Hansen et al. 2012b, Sims and Hansen In Press), but understanding Phytophthora 65 species in ecosystems is complex, and which species and why they occur is often unknown (Hansen 2012a).

Phytophthora species are grouped into 10 clades (clades = Greek klados or branches) from phylogenetic analysis, based on DNA sequence similarity in the ITS regions of the nuclear ribosomal genes (Cooke et al. 2000). Adding additional gene sequences to accompany ITS sequence data generally does not change a species’ clade association (Blair et al. 2008), suggesting that these relationships have an evolutionary basis that reflects natural relationships. The ten-clade system is now standard, and species are often described as belonging to a particular clade based on the ITS

Phytophthora phylogeny, and in some cases clades have been associated with particular life histories (Kroon et al. 2011). Three Phytophthora clades, clades 6, 7, and 2, were of particular concern.

Clade 6 Phytophthora species in general tend to be associated with forest streams. For example, Phytophthora gonapodyides (H.E. Petersen) Buisman has a cosmopolitan distribution and is documented from stream surveys around the world including Oregon stream surveys (Sutton et al. 2009, Hwang et al. 2009, Reeser et al.

2011, Jung et al. 2011). We expected that clade 6 species would be associated with stream water without necessarily being associated with alder disease. It has been proposed that clade 6 species are mainly aquatic and relatively scarce in forest soils 66

(Hansen et al. 2012a). However, some clade 6 species when given the opportunity can

act as aggressive tree pathogens (Durán et al. 2008), and others were recently found

associated with dying vegetation in Australia in areas where stream water had

inundated forest soils (Jung et al. 2011). Finding a clade 6 species in forest soils does not prove causation of disease but, if a typically aquatic species were common in soil, this would be evidence that an opportunity had arisen which transported it away from streams and into the rhizosphere (in the soil around roots) where it could act as a pathogen. Surprisingly, clade 6 P. gonapodyides, which is generally accepted as a ‘weak’ opportunistic pathogen only able to colonize non-suberized roots (Brasier et al. 1993)

has more recently been found to be, still opportunistic, but more aggressive than

previously supposed (Brown and Brasier 2007). In this study, we focused on forest

streams and soils that were riparian and surrounded alder tree roots. It was a goal to

examine the overall assemblage of Phytophthora species, with the objective of comparing species and clade composition from the stream water and the rhizosphere, and to compare species and clade composition from diseased alder roots. It was hypothesized that clade 6 species would be common in stream water. It was unknown if clade 6 species or any other species would be recovered from the rhizosphere or root systems, or if root systems would be diseased. Examining species and clade composition would help to understand not only clade 6 species, but also, in general, how species composition might be different from streams, the rhizosphere, and alder roots.

Clade 7 Phytophthora species in general are aggressive pathogens (Jung et al.

2002), and are responsible for many forest disease problems of hardwood trees. For

example, the invasive Phytophthora alni Brasier & S.A. Kirk is responsible for damaging

and killing about 15% of the alder across Southern Britain (Webber et al. 2004).

Phytophthora cambivora (Petri) Buisman in Italian chestnut (Castanea sativa Mill.)

forests is the causal agent of ink disease (Vettraino et al. 2001). Phytophthora

cinnamomi Rands in Southwestern Australia is the causal agent of Dieback of Jarrah

(Eucalyptus marginata Donn ex Sm.) and has a host list of around 1,000 species (Erwin and Ribeiro 1996, Robin et al. 2012) many of which occur in forests. Most but not all species in clade 7 have similar morphology. For example, species in clade 7 share the characteristics of having amphigynous, or both amphigynous and paragynous

attachment of the antheridia to the oogonial stalk, and being aggressive pathogens

(Jung et al. 2002). Phytophthora europaea E.M. Hansen & T. Jung, first recovered from

European oak forests, appears to be an exception. It was described as unique based on

having only paragynous attachment of antheridia and being a weak pathogen to

Quercus robur L. (Jung et al. 2002). Species in this clade are known to hybridize. For

example, P. alni is an emergent hybrid, soil and waterborne pathogen, causing a lethal

collar rot of alder in Europe. People have accidentally introduced the pathogen through

streamside planting of contaminated nursery stock (Jung et al. 2009). Once introduced

the pathogen spreads naturally with streams, floods, and drainage water, and the

pathogen negatively impacts natural alder stands (Gibbs et al. 1999). P. alni is now 68

present and its damage to alders is well documented in many countries across Europe.

The species is divided into three subspecies: alni, multiformis and uniformis. The most aggressive subspecies is P. alni subsp. alni Brasier & S.A. Kirk (Brasier et al. 2004). The less aggressive P. alni spp. uniformis Brasier & S.A. Kirk was recently found in the United

States in native forest soils in Alaska where it has not been found causing any apparent damage to the native thinleaf alder, Alnus incana (L.) Moench spp. tenuifolia (Nutt.)

Breitung (Adams et al. 2008). It was not known if any of the subspecies of P. alni were present in western Oregon riparian ecosystems, or if red alder, the most common alder species in western Oregon, would be impacted. Since Phytophthora cankers and canopy dieback on alder trees had been observed in western Oregon, it was of concern that P. alni could be causing the damage. It was discovered that another species, P. siskiyouensis, was the Phytophthora species recovered from Phytophthora bole cankers and it was suspected to be causing the disease (Chapter 1). However, it was of interest to evaluate other parts of the riparian alder ecosystem for P. alni to determine if it was present in streams, the rhizosphere, or alder roots.

Phytophthora clade 2 includes forest pathogens that have been recovered from

Oregon streams (Reeser et al. 2011), for example, Phytophthora pini Leonian,

Phytophthora plurivora T. Jung & T.I. Burgess, and Phytophthora siskiyouensis Reeser &

E.M. Hansen. P. pini was first described as a weak pathogen on the roots of Pinus

resinosa Aiton (Leonian 1925). It was later found that P. pini thrives in agricultural runoff 69

water (with a high pH around 9); and it may be a threat to the agricultural industry

where recycling water is a method of irrigation (Hong et al. 2011). P. pini had been

recovered in Oregon from areas with an agricultural influence (Hansen et al. 2012b), so

it was realistic to consider it would be recovered again if areas with an agricultural

influence were included in a survey. Another species recovered in Oregon, P. plurivora,

is a pathogen of many woody species including the European hardwood trees Fagus

sylvatica L., Quercus robur (Jung and Burgess 2009) and Alnus glutinosa. P. plurivora has

been found in association with alder trees in Europe (Alnus glutinosa) infected with the alder Phytophthora P. alni (Jung and Blaschke 2004, Jung et al. 2005 and Jung et al.

2009). P. siskiyouensis is a known alder pathogen that has been recovered from dying planted Alnus glutinosa L. (black alder) in Melbourne Australia (Smith et al. 2006) and from planted, dying Alnus cordata Desf. (Italian alder) in California (Rooney-Latham et al. 2007, Rooney-Latham et al. 2009). It is also known to occur in western Oregon where

P. siskiyouensis was first described after recovery from blighted Umbellularia californica

(Hook. & Arn.) Nutt. (myrtlewood) shoots and from Notholithocarpus densiflorus (Hook.

& Arn.) Manos, Cannon & S.H. Oh (tanoak) bark cankers in forests (Reeser et al. 2007).

In western Oregon, there had been no reports of P. siskiyouensis on alder, and no reports of P. siskiyouensis from alder from any natural ecosystems around the world.

In 2010, a survey, funded by the Forest Health Monitoring Program of the USDA

Forest Service, Pacific Northwest Region, began to examine dying alder trees along 70 streams in western Oregon (Chapter 1). Above ground symptoms, included

Phytophthora bole cankers, and P. siskiyouensis was isolated from this type of canker

(Chapter 1). It was unknown, however, if P. siskiyouensis was also associated with below ground symptoms in alder roots, or if P. siskiyouensis was associated with alders in general, from the rhizosphere or from stream water. Special care was taken to examine

Phytophthora species from the soil, roots, and stream water within riparian alder ecosystems. The goal in part, was to determine if P. alni and P. siskiyouensis were present, but also to determine and describe the assemblage of Phytophthora species present in these ecosystems. It was recognized that some species could be common in forests without necessarily being associated with disease (Hansen et al. 2012a, Oh et al.

2013, Sims and Hansen In Press). Following the 2010 alder survey an additional smaller survey was conducted by revisiting a subset of the 2010 established transects. The smaller coastal survey was done to determine the Phytophthora species recovered from surface sterilized diseased red alder roots.

The goal in this chapter is to describe the assemblage of Phytophthora species isolated and identified from riparian alder ecosystems in western Oregon. The specific objectives were to:

• Compare species and clade composition in the rhizosphere and in the stream

water; 71

• Compare species and clade composition from direct isolation of diseased woody

and fine alder roots;

• Evaluate for the presence of Phytophthora alni from all substrates;

• Determine if Phytophthora siskiyouensis or any other Phytophthora species was

consistently associated with the surveyed diseased alder stands or recovered

from diseased alder roots.

MATERIALS AND METHODS

The assemblage of Phytophthora species

Alder survey location and layout-Riparian alder stands in western Oregon (west side of the Cascade Mountains to the Pacific Ocean) were surveyed between 2 June

2010 and 19 October 2010, along 88, 100 m long by 10 m wide transects laid out lengthwise along streams (Chapter 1, Figure 1). Transects were not closer than 0.5 km linear distance from any other transect. Transects were selected to include alder trees exhibiting canopy dieback. Other selection criteria included access and bank stability. To assure reasonably uniform sampling across all of western Oregon, transects were divided among three sub-regions: the Willamette Valley sub-region with streams draining into the Willamette River (33 transects); the southern sub-region in SW Oregon

(25 transects), and the coastal sub-region with streams draining into the Pacific Ocean

(30 transects). All transects were in riparian alder forest but the amount of human disturbance in the surrounding landscapes differed greatly. One third of the transects in the Willamette Valley and coastal sub-regions were placed in forested landscapes, generally at higher elevations, and one-third were placed in downstream sites with suburban and rural residential development predominating in the surrounding landscape. The remaining sites were in intermediate locations, usually with agricultural land use adjacent to riparian areas. A subset of 18 transects were resampled in 2011

2012 in the coast sub-region. The resample focused on alder roots.

Sampling Phytophthora species from stream water and the rhizosphere-To

collect Phytophthora, two, one L water samples, five root samples, and two soil samples

were systematically collected per transect. Stream water was collected near alder roots

that extended into the water where these were present and/or beneath an alder

canopy, with one sample from near each end of a transect. Alder root samples were

collected near the base of alder trees (one sample/tree). Loose dirt was removed from

roots by shaking. Alder roots were identified by presence of root nodules or roots were

traced from the tree base. A soil sample was collected from two locations within each

transect, each beneath an alder tree with disease symptoms. Water, root, and soil

samples were brought back to the lab in a cooler and processed. Water samples were

filtered. Root and soil samples were baited with mature leaf pieces of Rhododendron.

All samples were plated onto oomycete selective media to target Phytophthora species.

Substrate: stream water - Water samples were divided into one 10 mL, two 50

mL and two 100 mL subsamples and were vacuum filtered onto five, 47 mm diameter 5

u nitrocellulose Millipore© filters per 1 L sample. Occasionally the amount that was

filtered was altered based on a priori knowledge about a particular area. If we were

expecting more Phytophthora from a site, then the amount of water filtered was reduced. If we were expecting less Phytophthora the amount of water filtered was

increased. This was done to maximize the number of plates with visibly separable

colonies. Funnel apparatus for filtration consisted of a two-piece glass system with 74

fritted glass base designed to handle the 47 mm diameter filter. Filters were inverted

and placed onto VARP+ media: 15 g Bactoagar, 50 mL V8 stock, 10 ppm rifamycin SV

sodium salt, 20 ppm Delvocid [50% natamycin salt], 200 ppm ampicillin sodium salt, 30

ppm Benlate [benomyl 50WP] with enough sterile water to make a slurry, and 50 ppm

hymexazol. Filters were incubated on the medium surface for 72 h at 20 °C. Filters were

removed and plates were examined for Phytophthora colonies using a dissecting

microscope at 20 x - 60 x variable magnification with trans-illumination.

For isolation, plates with one to 10 separable colonies visible after a 72 h incubation period were selected. Plates with overlapping colonies were discarded.

Visibly separable Phytophthora colonies were sub-cultured onto CARP media: cornmeal

agar, 10 ppm rifamycin SV sodium salt, 20 ppm Delvocid [50% natamycin salt], 200 ppm

ampicillin sodium salt (Reeser et al. 2011, Chapter 1). After an additional 48 - 96 h, filter

plates were checked again for slow emerging Phytophthora colonies and then again

after 10 or more days for slow to develop P. alni-like oogonia.

Substrate: rhizosphere (soil and unwashed alder roots) - One L soil and root

samples were collected in two L bags, and transported back to the lab in coolers. Roots

and soil were flooded with deionized water to approximately 3 cm above the soil/root

line. Flooding and baiting took place within 24 h of collection. Baits were made with

mature Rhododendron leaves grown in outdoor conditions. Strips, 2 cm wide centered 75 on the midvein were cut from leaves. Strips were then cut across the midvein into 2 mm wide pieces. Six bait pieces were placed in a tea bag paper with a foam packing peanut, stapled shut, and floated on the surface of the water over the flooded soil/roots. Baits remained in place for 72 h. Bait bags were removed and opened with forceps. The contents of one bait bag were placed on a clean paper towel. Leaf bait pieces were thoroughly blotted dry. Loose dirt was removed by wiping on the paper towel (Reeser et al. 2011). All clean, dry leaf pieces within each bait bag were partially submerged into

H/2 medium: cornmeal agar, 10 ppm rifamycin SV sodium salt, 20 ppm Delvocid [50% natamycin salt], 200 ppm ampicillin sodium salt, and 25 ppm hymexazol. Clean paper towels were used for processing each bait bag and forceps were dipped in 95% ETOH and flamed between each sample. Plates were checked again at 10 or more days for P. alni like oogonia.

Coastal subset survey location - A subset of 18 of the established 88 transects were revisited in December 2011 through March 2012 to follow-up on tree symptoms, especially bleeding cankers, and the root cankers. Red alder was the only alder tree species sampled. Five alder trees from each transect were sampled. Five root samples and five above ground bark samples were systematically collected per transect. Samples were brought back to the lab in a cooler and processed. Bark methods and results are presented in Chapter 1.

Sampling Phytophthora species from alder roots - Root samples were washed to remove soil, soaked under running water for 3 h, and drained. 500 mL of 10 % bleach

(0.6 % sodium hypochlorite) was added to the root sample in a bag and shaken for 30 s.

Root samples were rinsed to remove bleach, and soaked under running water for 10 minutes. Root samples were divided into fine and woody root pieces. Woody roots were defined as roots with an outer bark layer. Fine roots were roots with a thin outer epidermal layer and were < 2 mm. Each root type was examined for cankers, necrotic spots, and/or water soaked lesions. Symptomatic pieces of roots of each root type were directly plated onto H/2 media.

Phytophthora species determination - Morphological characterization to genus was done on culture plates. Morphological characters used to distinguish Phytophthora species from other fungi growing on the plates included rate of growth, branching pattern and greater refraction of light by hyphae in media when compared to Pythium species. Occasionally isolates were ambiguous (it was uncertain if they were

Phytophthora or another oomycete such as Pythium). Ambiguous isolates were grown in water culture to examine zoosporogenesis. If cytoplasm from an isolate was observed to move from the sporangium into a vesicle prior to zoospore cleavage then the isolate was classed ‘not Phytophthora’ and discarded. If cytoplasm from an isolate was observed to cleave within sporangia, typical of Phytophthora zoosporogenesis, then isolates were grouped with Phytophthora. For a few isolates, zoosporogenesis did not 77 occur and these isolates were classed as uncertain identity and set aside for DNA extraction.

All isolates classed as Phytophthora or uncertain identity were DNA extracted using a DNeasy Tissue Extraction Kit (Qiagen Inc., Valencia, CA). PCR was performed in a

50 μl reaction containing 1 μl template DNA. PCR product was sequenced using the mitochondrial Cox spacer region flanking the Cox1 and Cox2 genes (Martin & Tooley

2003). Sanger sequencing was done by Oregon State University Center for Genome

Research and Biocomputing sequencing center. Each isolate was sequenced in one direction with either FMPHy-8 or FMPHy-10. Sequences were aligned in Clustal X2

(Larkin et al. 2007). Species were determined by matching sequences to a known reference sequence from the Hansen Phytophthora laboratory at Oregon State

University (HPLOSU). If an isolate did not match a reference isolate 100%, then it was sequenced in the opposite direction. If it still did not match then DNA was PCR amplified and sequenced in the internal transcribed spacer region (ITS) with primers DC6 and ITS4

(Cooke et al 2000). Amplified products were then sequenced with DC6, ITS2, ITS3 and

ITS4 as sequencing primers (White et al 1990). Contigs were assembled and the overlapping sequences were manually edited using an editing package (Staden 1996).

Edited sequences were aligned with ITS reference sequences of forest Phytophthora species recovered in Oregon and available for comparison at HPLOSU. If an isolate’s

DNA still did not match a reference then it was grouped with the closest reference 78 species based on clustering in a phylogeny and manual examination of base pair differences in the alignment. Information about isolate reference match, species identification, location of recovery and sample type were recorded in a database.

Data analysis – From the alder survey sampling efforts species/substrate associations were compared. To compare isolates from different substrates the number of isolates for a species was weighted based on the total number of isolates recovered from each substrate. Then, the data were normalized for each species to rescale the numerical weighted count between zero and one:

Sι,0_το_1 = (Sι − Smin )/(Smax − Smin )

Where: Si = the count for weighted speciesi, Smin = the minimum possible count for a species, Smax = the maximum count from all substrates within the speciesi, Si, 0 to 1 = the speciesi count data normalized between 0 and 1.

After weighting and normalizing the data, associations were assigned if the (Si) proportion of the data from a species was > 0.5 for a particular substrate and a goodness of fit test, based on the weighted count, suggested a particular association at a 95% confidence level. Species associations and the goodness of fit test were only 79 examined if the number of isolates for a species were at least five from each substrate, because the true sampling distribution of the goodness of fit statistic is approximately chi-squared and the chi square approximation can be a poor predictor for sparse counts

(Agresti 2007).

RESULTS

The assemblage of Phytophthora species

Combined survey details

Overall assemblage - Phytophthora species were recovered from 83 of the 88

transects sampled in western Oregon riparian ecosystems in association with alder

trees. Three Phytophthora species were recovered from all substrates (stream water,

rhizosphere, woody roots and fine roots): P. gonapodyides (clade 6), P. taxon

Pgchlamydo (clade 6), and P. siskiyouensis (clade 2, Table 1 gray rows). In total 20

Phytophthora species from eight clades were recovered (Table 1). Most of the species

recovered were from clades 2 (four species), 6 (six species), and 7 (four species). Two

species were recovered from surface sterilized roots that were not recovered from

water or rhizosphere sampling; these were P. alni spp. uniformis and P. europaea. The other six species recovered were from clades 1,3,8,9 and 10.

Incidence of Phytophthora alni, and location details - Phytophthora alni spp. uniformis was recovered (isolates: 110-R-1N.1, 118-R-1K.1, 118-R-1J.3, 118-R

1101711.4) from necrotic lesions on surface sterilized woody roots of two red alder

trees from the coastal subset survey. Recovery was from two transects, one transect

alongside the Smith River in Douglas County, and one transect alongside Cape Creek in

Lane County. In addition, an isolate from the Smith River in Douglas County, identified

as P. cambivora based on ITS sequence and morphology matched P. alni spp. alni in the 81

Cox spacer region. This P. cambivora isolate (112-R-1O.2) and an unknown species closest to P. europaea (112-R-1O.3), but with unique morphology, were recovered from the same alder tree root system, and downstream from a tree that yielded P. alni spp. uniformis. Phytophthora siskiyouensis was recovered (110-B-4N.1) from a tree with bark cankers from the same transect along the Smith River from which P. alni spp. uniformis was recovered from woody roots (110-R-1N.1). Both P. gonapodyides and P. taxon

Pgchlamydo were recovered from woody roots of trees located along the Smith River as well. Prior to root discovery, P. alni was not recovered from any substrate (rhizosphere or stream water).

Table 1) Phytophthora species recovered from different substrates in western Oregon riparian ecosystems. Three species were recovered from all substrates (gray rows)

Presence or absence substra te † woody fine Phytophthora species ITS Clade roots roots rhizosphere water P. nicotianae 1 - - - + P. citricola 2 - - - + P. pini 2 - - - + P. plurivora 2 - - + + P. siskiyouensis 2 + + + + P. pluvialis 3 - - - + P. pseudosyringae 3 - + - + P. gonapodyides 6 + + + + P. gregata 6 - - + + P. lacustris 6 - + + + P. riparia 6 - - - + P. taxon Oaksoil 6 - - + + P. taxon Pgchlamydo 6 + + + + P. alni spp. uniformis 7 + - - - P. cambivora 7 + + + - P. cinnamomi 7 - - + - P. europaea 7 + - - - P. cryptogea 8 - - + - P. parsiana 9 - - + + P. gallica 10 - - + + total species 20 6 6 12 15 total clades 8 3 4 6 6 † all substrates are associated with alder, roots are from red alder

Alder Survey

Phytophthora species assemblage from stream water and the rhizosphere - A total of 1190 Phytophthora isolates representing 18 species were recovered from 83 stream water and the rhizosphere in the alder survey conducted across western Oregon

(Table 2). Recovered species were from eight of the 10 ITS clades, including all clades except clades 4 and 5. Most isolates (> 50%) and most species (15) were recovered from filtered stream water (Table 1). The number of recovered isolates was 3.8 times greater

(based on the number of recovered isolates) from stream water than from the rhizosphere. After equivocal weighting, normalizing and examining the goodness of fit for a particular association, two species were associated with both substrates, three species were associated more with the rhizosphere, and one species was associated more with stream water (Table 2). Most of the species were not assigned an association because they were recovered from one or more of the substrates less than five times

(Table 2). 84

Table 2) Substrate associations from western Oregon riparian ecosystems Proportion of isolates† Phytophthora species ITS clade Rhizosphere Stream water Number of isolates Goodness of fit p-value rhizosphere and stream water P. siskiyouensis 2 0.6 0.4 17 0.32 P. parsiana 9 0.5 0.5 25 1 rhizosphere P. gonapodyides 6 0.8 0.2 267 <0.0001 P. lacustris 6 0.7 0.3 136 <0.0001 P. taxon Pgchlamydo 6 0.7 0.3 63 0.0007 stream water P. taxon Oaksoil 6 0.1 0.9 506 <0.0001 unknown‡ P. nicotianae 1 0.0 1.0 2 na P. citricola 2 0.0 1.0 1 na P. pini 2 0.0 1.0 16 na P. plurivora 2 0.2 0.8 20 na P. pseudosyringae 3 0.0 1.0 111 na P. pluvialis 3 0.0 1.0 1 na P. gregata 6 0.9 0.1 3 na P. riparia 6 0.0 1.0 2 na P. cambivora 7 1.0 0.0 10 na P. cinnamomi 7 1.0 0.0 4 na P. cryptogea 8 1.0 0.0 1 na P. gallica 10 0.9 0.1 5 na total species 12 15 18 total isolates 247 943 1190 † weighted and normalized, ‡ no association was assumed

Species and clade composition from stream water – The filtered stream water was composed of 943 isolates and 15 species from six clades (Table 2). The clade 6 species composed 81% of the isolates from stream water. Phytophthora taxon Oaksoil, a clade 6 species, composed 52% of the recovered isolates (Figure 1). The other three clade 6 species (that composed at least 1% of isolates) recovered were P. gonapodyides

(16%), P. lacustris (9%) and P. taxon Pgchlamydo (4%). Three species from clade 2 85

composed 5% of the recovered isolates (P. pini, P. plurivora, and P. siskiyouensis). P.

pseudosyringae (clade 3) composed 12% of the recovered isolates. P. parsiana (clade 9)

composed 2% of the recovered isolates. Species that composed < 1% of the recovered

isolates from stream water included: P. nicotianae, P. citricola sl, P. pluvialis, P. gregata,

P. riparia, and P. gallica (other, Figure 1).

Species and clade composition from the rhizosphere - The rhizosphere sampling

included isolates recovered from soil and root baits. There were no meaningful

differences between the species composition from soil or root baits so they were

combined as rhizosphere sampling. A total of 247 Phytophthora isolates, representing

12 species from seven clades were from the rhizosphere (Figure 1b). The isolates from

the rhizosphere were mostly species in clade 6 (88%). P. gonapodyides accounted for

49% of the recovered isolates. The other clade 6 species recovered were P. lacustris

(21%), P. taxon Pgchlamydo (10%) and P. taxon Oaksoil (8%). Clade 2 P. siskiyouensis

composed 2% of the recovered isolates from the rhizosphere. Clade 7 species (P.

cambivora and P. cinnamomi) composed 6% of the recovered isolates. Clade 9 P.

parsiana composed 2% of the recovered isolates. Clade 10 P. gallica composed 1% of

the recovered isolates. The other species recovered from the rhizosphere that

composed 1% or less of the isolates were: P. plurivora, P. gregata, and P. cryptogea.

(a)

Key Color Clade Rhizo Water 2 3 NA 6 7 NA 9 10 NA

(b)

Figure1) Phytophthora species composition from (a) stream water and (b) the rhizosphere associated with alder in western Oregon riparian ecosystems. Recovery was from 88 transects.

Comparison of Phytophthora from streams and the rhizosphere - Clade 6 species

composed the majority of isolates from both stream water and the rhizosphere in

similar proportions (81% and 88% respectively). The clade 6 species recovered from

both substrates were the same: P. gonapodyides, P. lacustris, P. taxon Oaksoil, and P.

taxon Pgchlamydo (Figure 1a-b). However, P. taxon Oaksoil composed the majority of

isolates from stream water (52% of all Phytophthora from stream water) and P. gonapodyides composed the majority of isolates from the rhizosphere (49% of all

Phytophthora from the rhizosphere). Clade 2 P. siskiyouensis was present from both substrates, and so was clade 9 P. parsiana. All other species were only present in either one or the other substrate. Clade 7 and clade 8 species were not recovered from stream water but were recovered from the rhizosphere. Clade 3 species P. pseudosyringae and

P. pluvialis were not recovered from the rhizosphere but were recovered from stream water.

Coastal subset survey

Phytophthora species and clade composition from red alder fine roots - Six species (37 isolates) in four clades were recovered from fine surface sterilized roots

(Figure 2, Figure 3 F). The majority of isolates recovered from fine roots were clade 6 species (76%), including P. gonapodyides (43 % of all fine root isolates), P. lacustris (3%),

and P. taxon Pgchlamydo (30%). One species was recovered from each of the remaining 88 three clades: P. siskiyouensis (11%, clade 2), P. pseudosyringae (8%, clade 3), and P. cambivora (5%, clade 7).

(a)

Key Color Clade Fine Woody 2 3 NA 6 7

(b) P. siskiyouensis 4% P. europaea P. cambivora 4% 9% P. alni spp. uniformis 9%

P. P. taxon gonapodyides Pgchlamydo 52% 22%

Woody roots number of isolates = 23

Figure2) Phytophthora species composition from (a) fine roots and (b) woody roots of red alder from western Oregon riparian ecosystems. Recovery was from 18 transects.

Phytophthora species and clade composition from red alder woody roots - From necrotic lesions on woody surface sterilized roots (Figure 2, Figure 3 W) 23 isolates, six species in three clades were recovered. Most isolates recovered from woody roots were clade 6 species (74%). Two clade 6 species were recovered: P. gonapodyides (52%), and

P. taxon Pgchlamydo (22%). Three species from clade 7 were recovered: P. alni (9%), P. cambivora (9%), and P. europaea (4%). The remaining species recovered was (clade 2) P. siskiyouensis (4%).

Figure 3) Diseased alder root piece that recovered Phytophthora species from both fine and woody root necrosis. (F) Fine root necrosis (W) woody root necrosis.

Comparing species and clade composition from fine and woody roots - Clade 6 P. gonapodyides and P. taxon Pgchlamydo composed most of the isolates recovered from both fine and woody roots. Clade 7 species were more often recovered from woody than fine roots and composed a greater proportion of isolates from woody roots than from any other substrate. P. alni spp. uniformis was recovered from woody roots and P. cambivora was recovered from both root types. Clade 2 P. siskiyouensis was recovered from both root types.

DISCUSSION

The assemblage of Phytophthora species

Combined survey details

The overall assemblage – Phytophthora species were widespread in association with alder trees from western Oregon riparian ecosystems. Most transects (83 out of

88) recovered Phytophthora species, and twenty different species were recovered.

However, only three out of the twenty species were recovered from all substrates, which were diseased root pieces (both woody and fine), the rhizosphere around diseased trees, and from stream water associated with alder trees (Table 1). Only red alder roots were examined, other Phytophthora species might be associated with white alder tree root disease. In addition, all three species were recovered from locations across western Oregon. All three of the species were from clades that were of particular concern in our survey (clades 2, 6 and 7). Two species were from clade 6 and one species was from clade 2.

The three species from clades of particular concern that were recovered from all substrates were: P. siskiyouensis (clade 2), P. gonapodyides (clade 6), and P. taxon

Pgchlamydo (clade 6). It was an objective to determine if Phytophthora siskiyouensis or any other Phytophthora species was consistently associated with the surveyed diseased alder stands or recovered from diseased alder roots. The evidence provided on the 93

assemblage of Phytophthora species, suggests these three species, which includes P.

siskiyouensis, were consistently recovered around and from diseased alder trees. Little is

known about P. taxon Pgchlamydo which is not a formally described species; there is recent evidence this species can hybridize with other species (Burgess 2012), which may

make it difficult to formally describe. Our evidence suggests it is a candidate root

pathogen because 30% of the fine root isolates and 22% of woody roots isolates were

composed of this species. P. gonapodyides and P. siskiyouensis are the other two

species, and are known pathogens of alder. When P. gonapodyides was first described

as Pythiomorpha gonapodyides H.E. Petersen; one of the three places it was recovered

from was an alder tree in Denmark (Petersen 1910). About the same time that P.

siskiyouensis was being described as a species from bark cankers on tanoak trees in

Oregon (Reeser et al. 2007) it was being described as a pathogen of alder in Australia

(Smith et al. 2006) and California (Rooney-Latham et al. 2009). These survey efforts

present the first evidence of these two species (P. gonapodyides and P. siskiyouensis) as pathogens of alder in Oregon, and in natural ecosystems in the United States. In fact, P. siskiyouensis has not been described as a pathogen of alder in natural ecosystems anywhere in the world. The first evidence that P. siskiyouensis was a pathogen of alder in natural ecosystems in western Oregon was presented in Chapter 2; there, P. siskiyouensis was described after being recovered from Phytophthora bole cankers. A population genetics study would be the most appropriate method of determining if P. siskiyouensis is a native pathogen to the Pacific Northwest. 94

Evaluation for P. alni – Phytophthora alni spp. uniformis was recovered from

necrotic red alder roots, but not from any other substrate or alder species. Before this

survey there was fear that the invasive species P. alni was causing Phytophthora cankers

on alder in western Oregon riparian ecosystems. We did not recover P. alni spp. uniformis from Phytophthora cankers on the boles of alder. The simplest answer is that

Phytophthora siskiyouensis is the only species responsible for Phytophthora bole cankers in western Oregon USA at this time. However, other Phytophthora species including P. alni spp. uniformis and other clade 6 and clade 7 Phytophthora species may be responsible for root disease. It is also possible that other Phytophthora species could be causing bole cankers on alders and just have not yet been isolated.

Comparing situations - There were differences between the Oregon situation and the Alaskan situation. For one, survey efforts focused on different alder species. The

Alaskan alder survey focused on thinleaf alder and only examined thinleaf alder. This survey focused on red alder and examined red alder, white alder and to a lesser extent thinleaf alder. The habit of alder that was examined between the two surveys was different. The Alaskan alder survey examined shrubs and this survey examined only trees. The results were also different. In Alaska there was no apparent root disease on the examined thinleaf alder shrubs. In Oregon there was apparent root disease on the red alder and white alder trees. In Alaska P. alni spp. uniformis isolates were recovered 95 from remote locations baited from around healthy root systems of thinleaf alder shrubs.

In Oregon P. alni spp. uniformis isolates were recovered from locations with a history of human use from direct isolation from diseased red alder roots.

There were also similarities in the Alaskan situation and the Oregon situation.

Both states contain red alder and thinleaf alder. To our knowledge the red alder from

Alaska has not been examined. In Oregon, only two isolates of P. gonapodyides were recovered in association with transects containing thinleaf alder and those isolates were only from stream water. No other Phytophthora species or isolates were recovered in association with thinleaf alder in Oregon and no isolates were recovered directly from thinleaf alder, suggesting these isolates may have even been incidental to the site. In our study and in the Alaska study thinleaf alder did not appear to be having a

Phytophthora root disease problem. If we consider it on a tree to tree basis, based on the number of trees examined in Oregon, we had a 1% chance of finding a Phytophthora species isolate in association with a thinleaf alder tree, whereas from red alder we had a

71% chance of recovering a Phytophthora species isolate. It is possible that alder species differences could be important.

Other species - The list of Phytophthora species from this survey was very similar to an earlier stream survey in western Oregon (Reeser et al. 2011). However, this stream survey recovered five additional species: P. citricola sl, P. gallica, P. gregata, P. 96 nicotianae, and P. parsiana. Identity of each was initially determined by Cox spacer sequence, then confirmed by morphological examination and additional sequencing in the ITS region. The survey design may have facilitated the recovery of these species.

Transects were laid out along a stream based on human impact level and western

Oregon was divided into three sub-regions in order to adequately represent riparian ecosystems (see methods). At this time there is little evidence these species are pathogens of alder; however, the fact they are likely introductions to riparian forest ecosystems in Oregon may be of interest.

Two of the species, the species in the P. citricola complex, and P. nicotianae were recovered only from high impact level transects and P. parsiana, P. gallica and P. gregata were all recovered from high and mid impact level transects. If we had placed transects in only low impact areas, there is no evidence these species would have been recovered. In addition, a species richness analysis suggested greater species richness at higher impact levels (appendix).

P. parsiana is an agricultural pest of woody plants around the world. Woody plant hosts and locations of recovery included in the species description paper were pistachio (Pistacia vera) from Iran and the US, fig (Ficus carica) from Iran, and almond

(Prunus dulcis) from Greece (Mostowfizadeh-Ghalamfarsa et al. 2008). Since its description in 2008 several new woody hosts have been described (Rafiee and 97

Banihashemi 2013). The isolates from Oregon fall closest to P. parsiana and so are

grouped with this species, although the isolates from Oregon may be part of a species

complex within P. parsiana and P. hydropathica; this group should be reexamined taxonomically using isolates recovered from Oregon. P. parsiana is a recently described species with very little known about its distribution and complete host range, it was only recovered from sites with considerable human influence so it is a likely introduction to

Oregon, possibly from streamside planting, possibly from one of the various Prunus species or Ficus species which are commonly planted and sold in Oregon. P. parsiana

was recovered from the lower portions of: the Smith River in Douglas County, the coast

fork of the Willamette in Lane County, the Alsea River in Lincoln County, from Jackson

creek where it runs through a nursery in Jackson County, and from the Rogue River from

a location with obvious agricultural influence.

P. nicotianae was first described as a tobacco pathogen (van Breda de Haan

1896), but the species is accepted as being plurivorous and contains many woody plant hosts (Hall 1993), some of which are ornamentals planted in Oregon. Examples of woody plant hosts grown in Oregon include Magnolia stellata and Rhododendron

‘Mikkeli’ (Schwingle et al. 2007). The isolates we have do not match P. nicotianae in the

ITS region exactly (or any isolates in the Gen-bank repository); however, our isolate is within the boundaries of morphological characters of P. nicotianae. In the past P. nicotianae encompassed different species and varieties within those species but these 98

were regrouped as a single species because evidence did not support splitting (Hall

1993). Our isolates may have entered the streams because they were washed from a

streamside planting of a host plant species.

Our P. citricola isolates do not match P. citricola ss in the ITS region, but our

isolates are within the P. citricola complex (Bezuidenhout et al. 2010) and are closest to

P. citricola ss in an ITS alignment. Our isolates differ in ITS by a single base pair and an

insertion from P. citricola ss. They also differ from a taxon called P. citricola E in Jung

and Burgess (2009) by two base pairs. P. citricola E was P. citricola in the seminal 2000

description of the genus Phytophthora based on ITS (Cooke et al. 2000). It is accepted

this is not P. citricola ss (Jung and Burgess 2009). P. citricola E is also referred to as P.

citricola clade E in Bezuidenhout et al. (2010). However, P. citricola E has not been

redefined as a new species. We refer to our isolates that, in the ITS region, are between

P. citricola E and P. citricola ss, as P. citricola sl within the P. citricola species complex.

Our isolates are closer in the examined gene region to P. citricola than to other species within the citricola complex such as P. pini or P. plurivora.

P. gregata has been described from isolates recovered from declining vegetation in Australia (Jung et al. 2011). P. gregata had not been reported in the United States before this survey. There is evidence for hybridization of P. gregata with P. taxon

Oaksoil. The putative hybrid was sequenced in the B-tubulin region and the isolate had 99

20 double peaks, each peak corresponding exactly to one of the two different parent

species (Table 3). The high peak in the sequence calls always corresponded to the P.

gregata isolate from Australia (GB JN547605) and the lower peak always corresponded

with P. taxon Oaksoil. P. gregata has not previously been reported in the United States.

It could be hybridizing with the common aquatic species in streams, P. taxon Oaksoil, in

Oregon. The isolates of P. gregata recovered in Oregon do not match (100%) any other

isolates recovered anywhere else in the world in the ITS and Cox spacer regions.

Table 3) Base pairp chart of Beta -tubulin p double peaks

P. taxon Oaksoil T C T C C C A T T C C T C A G A A C T C Putative hybrid low peak T C T C C C A T T C C T C A G A A C T C Putative hybrid high peak C T C G T G G C G T A G T G A G G T C T P. gregata JN547605 C T C G T G G C G T A G T G A G G T C T POSITION 31 34 40 46 49 115 127 163 187 190 316 325 364 382 430 445 481 673 718 745 The sequence calls are split in the table to display high and low double peaks. The high peak always corresponded with P. gregata and the low peak always corresponed with P. taxon Oaksoil.

P. gallica was isolated from two locations along the Coast Fork of the Willamette

River in Lane County Oregon. Two of the isolates were recovered (beneath different

trees) from a transect that is adjacent to a city park near where Interstate Highway 5

crosses over the stream. This is a site from which P. siskiyouensis was recovered from white alder trees with large cankers and planted red alders were observed to have

Phytophthora cankers. P. gallica was also isolated from an upstream location adjacent to a park. There was genetic variability in all recovered isolates. One matched exactly to 100 an isolate from Australia in the ITS region (GB DQ286726). The other two did not match

(100%) to any isolates in the Gen-bank repository, and were different from each other.

This is the first documented case of P. gallica in the US outside of Alaska (Adams et al

2009).

P. europaea recovered from this study matched another Gen-Bank isolate from

Oregon (HM004226 , Reeser et al. 2011) in the ITS gene region, but were different from isolates recovered from Europe (AF449493, Jung et al. 2002) and from isolates recovered in Eastern and North-Central US (DQ313222, Balci et al.2006). Our Oregon isolates also differed from the description of the species (based on the European type isolate, Jung et al. 2002) in the attachment of the antheridia to the oogonial stalk. The isolates recovered from Oregon in this study had both paragynous and amphigynous attachments (Figure 4) of the antheridia to the oogonial stalk. Having both paragynous and amphigynous attachments is a characteristic of all other species within clade 7 except P. europaea (Jung et al. 2002). This species was uncommon, but recovered from diseased alder roots. This species was recovered from the same stand of alders along the Smith River in Douglas County from which P. alni spp. uniformis was recovered.

101

Figure 4) P. europaea from western Oregon with amphigynous attachment of the antheridium to the oogonium stalk. Bar 50 μ

Alder survey

Comparing species and clade composition from stream water and the

rhizosphere - Clade 6 Phytophthora species have been proposed to belong to a guild of

aquatic opportunistic pathogens(Hansen et al. 2012 a). We found that clade 6

Phytophthora species were common from streams, composing 81% of stream isolates.

However, clade 6 species were also as common from the rhizosphere (88% of

rhizosphere isolates) of riparian soils. P. gonapodyides and P. Pgchlamydo were the

most frequently recovered species from diseased alder root tissue (Figure 2a-b). P.

taxon Oaksoil was the most prevalent in streams (52% stream isolates) but only

composed 8% of the rhizosphere isolates, and was absent from root recovery. This 102

habitat switching occurred for P. gonapodyides as well (49% of the total rhizosphere

isolates were P. gonapodyides, Figure 1b compared to 16% of the total stream water isolates Figure 1a). In addition, isolates of P. gonapodyides, and P. taxon Pgchlamydo recovered from this survey were able to cause significant lesions to red alder in summer stem inoculation trials, winter stem inoculation trials, and zoospore root dip inoculations (Navarro 2013). In the same pathogenicity tests P. taxon Oaksoil was not able to cause lesions larger than controls (Navarro 2013). Alder has been reported as a host of P. gonapodyides in the past (Petersen 1910, Erwin and Ribeiro 1996) and, P. gonapodyides, first described as Pythiomorpha gonapodyides H.E. Petersen, was recovered from alder (Petersen 1910). In this study, we found that P. gonapodyides was associated more with the rhizosphere than from stream water (Table 2) focused on forest soils surrounding diseased alder tree roots. In flooded forest soils clade 6 species that survived the move from stream water to the rhizosphere could have access to root systems of trees in riparian areas. There is strong evidence Phytophthora gonapodyides is an alder root pathogen in western Oregon. How important this is to alder tree health is not known, and it is not known if the disease we were observing is new, or if it has just been missed.

Coastal subset survey

Comparing species and clade composition from fine and woody roots - Clade 6 P. gonapodyides and P. taxon Pgchlamydo were the most frequently recovered species 103

from both fine and woody roots. P. taxon Pgchlamydo was more common from surface- sterilized roots than from either the rhizosphere or the stream water, supporting evidence that it me be a red alder root pathogen. P. gonapodyides was about as common from roots as it was from the rhizosphere. Alder has been reported as a host of

Phytophthora gonapodyides in the past (Petersen 1910, Erwin and Ribeiro 1996) and it is a likely pathogen of alder in western Oregon. Clade 7 species were more common from woody than fine roots and were more common from roots than from any other substrate. P. cambivora, P. siskiyouensis, P. gonapodyides and P. Pgchlamydo were recovered from both root types. These Phytophthora species may be important in alder root disease.

Species determination

Molecular methods – Identifying large numbers of Phytophthora isolates is challenging. We relied initially on DNA sequence matching of a one-way read of the mitochondrial Cox spacer region with previously identified reference isolates. The sequenced region is relatively short (approximately 260 bases), and the method is relatively fast. Although the Cox spacer is more variable than Cox I or II or ITS (Martin and Tooley 2003, Schena and Cooke 2006), in nearly all cases, Cox spacer identifications matched ITS-based identifications (Martin and Tooley, Schena and Cooke 2006), if an adequate set of reference sequences capturing the range of variation was available. We found that intraspecies variation within the Cox spacer region was common among the 104

20 Phytophthora species identified in this study. In total, there were 47 Cox spacer

genotypes (Table 4). Species were identified because they grouped in a terminal clade

(in a phylogeny) with a particular reference species. Usually, isolates that did not match

a reference in Cox spacer, did not match a reference in ITS (Table 4). However, when isolates did not match in the Cox spacer and ITS they still clustered with the same species for both regions, with one exception (Table 4 P. cambivora Isolate: 112-R-1O.2).

The one exception was for one isolate that matched P. alni spp. alni in the Cox spacer region; but it was more similar to P. cambivora, in that it was heterothallic and in the ITS region it matched P. cambivora. Since isolates that did not match in the Cox spacer and

ITS regions, still clustered with the same species, this suggests that much of the additional variability in Cox spacer represented variability in the ITS region.

105

Table 4) Phytophthora species recovered in western Oregon riparian ecosystems and reference matches. Reference match

HPL OSU GenBank

Species ITS clade isolate Cox spacer ITS Cox spacer ITS P. nicotianae 1 207-W-2.4 none none none none P. citricola sl 2 15-W-1.5 III 5100B1F none none none P. pini 2 112-W-1.1 V 4-3P - - HM004227 P. plurivora 2 3-W-1.34 151.77 - - - P. plurivora 2 121-W-1.12 none none none none P. siskiyouensis 2 222-29-B.1 33-2-3.2-1102 WA5-030403 - EF23386 P. siskiyouensis 2 113-W-2.16 118-R-1081011.5 none none none P. pluvialis 3 19-W-2.3 none WA28-022404 KC853447 HM004217 P. pseudosyringae 3 120-W-1.11 33-2-3.1-1102 - - - P. pseudosyringae 3 102-W-1.1 WA11-111302 - - - P. pseudosyringae 3 125-W-2.12 WA64.2-080304 - - - P. pseudosyringae 3 113-W-1.12 WA1.2-021903 - - - P. pseudosyringae 3 120-W-2.20 none none none none P. pseudosyringae 3 117-W-2.8 none WA1.2-021903 - - P. gonapodyides 6 115-8-R.1 AB4 - - - P. gonapodyides 6 218-41-R.1 none WA21-011304 - - P. gonapodyides 6 31-1-S.3 none WA21-011304 - - P. gonapodyides 6 31-1-S.2 none WA21-011304 - - P. gregata 6 11-3-R.1 none none none none P. lacustris 6 107-W-2.8 WA21-091603 - HM004219 P. lacustris 6 33-2-R.1 none none none none P. lacustris 6 33-2-R.6 none none none none P. riparia 6 208-W-2.6 SOC2.7.29 SOC2.7.29 JQ626581 JQ626594 P. riparia 6 104-W-1.16 VI 3-100B9F - - HM004225 P.riparia 6 33-W-2.1 none none none none WA46.3-101804 & P. taxon Oaksoil 6 101-W-1.3 IV_3-100A1F - - HM004234 P. taxon Oaksoil 6 101-W-1.1 VI 5-100B1F - - - P. taxon Oaksoil 6 108-W-1.15 107-W-2.3 - - - P. taxon Oaksoil 6 219-W-1.1 none none none none P. taxon Oaksoil 6 122-W-2.12 none none none none P. taxon Oaksoil 6 123-W-1.2 none none none none P. taxon Pgchlamydo 6 104-W-1.14 133 - - - P. taxon Pgchlamydo 6 113-W-1.16 WA5.1-072003 - - HM004224 WA46.3-100404 & I 5 P. taxon Pgchlamydo 6 102-2-R.1 200A1F - - - P. alni spp. uniformis 7 118-R-1K.1 AK53.5 none none none P. cambivora 7 112-R-1O.2 4048.2† none DQ162846†† - P. cambivora 7 111-R-4O.1 WA18.1-111003 - - none P. cambivora 7 112-R-2O.1 WA18.1-111003 4048.2 - EF486693 P. cinnamomi 7 223-2-R.1 9641.1 - - - P. europaea 7 112-R-10.1 none VI 1-2P - HM004226 P. cryptogea 8 33-2-S.2 MRW2.3.11A - - - P. parsiana 9 207-W-2.6 RWC2.7.8B - - - P. parsiana 9 111-2-R.1 WA23.3-081803 - - - P. parsiana 9 33-2-R.5 none none none none P. gallica 10 33-14-S.1 none none none DQ286726 P. gallica 10 33-4-R.1 none none none none P. gallica 10 32-W-2.4 none none none none † P. cambivora. Note: Morphology of P. cambivora †† P. alni spp. alni. Reference: Schena and Cooke 2006 106

CONCLUSIONS

• Phytophthora species are widespread and diverse in association with alder in

western Oregon.

• However, only three species were associated wit h all substrates: clade 2 P.

siskiyouensis, and clade 6 P. gonapodyides and P. taxon Pgchlamydo.

• Phytophthora alni spp. uniformis was recovered, but only from necrotic red alder

roots of two trees.

• P. taxon Oaksoil was the most common species but mainly from stream water,

rarely from the rhizosphere, and not from alder roots.

107

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Chapter 4. Pathogenicity of Phytophthora siskiyouensis, P. alni subspecies uniformis and P. taxon Oaksoil to alder trees in western Oregon

INTRODUCTION

Reports of alder (Alnus Miller (Betulaceae)) trees with canopy dieback in riparian

ecosystems have increased in recent years in western Oregon. Concerns about the

health of alder were heightened by recent disease problems in other parts of the world,

especially the lethal root and collar disease of alder in Europe caused by Phytophthora

alni Brasier & S. A. Kirk. In 2010, surveys of alder trees began in western Oregon to

identify possible causal agents of the observed damage. The surveys focused on alder

stands exhibiting canopy dieback and special efforts were made to catalog associated

Phytophthora species. Stands were examined for bole and root symptoms on alder trees

similar to those caused by P. alni in Europe. Phytophthora species were collected and

identified from symptomatic alder tissues, as well as from leaf debris, rhizosphere soil,

and adjacent stream water. In this chapter, we summarize the association of three key

Phytophthora species with declining (cankers/canopy dieback) alder and test their

pathogenicity. The goal is to complete Koch’s Postulates to test a causal relationship

with cankers, and to test an association with alder canopy dieback for the two most

prevalent species. 115

Phytophthora species are Oomycetes, water molds, in the Peronosporales order and Peronosporaceae family (Hulvey et al. 2010, Thines 2013). While they have some morphological similarities to Fungi including hyphae and spores, for example, they are unrelated to true Fungi. Most, if not all, species of Phytophthora are plant pathogens.

Characteristic symptoms of Phytophthora species infecting hardwood trees include phloem island lesions (surrounded by a dark outer boundary (Brown and Brasier 2007).

To successfully isolate the causal agent from the diseased host rigorous sampling is often necessary, because of faster growing microorganisms that might share the tissue, and host phenolic compounds that inhibit the growth of some Phytophthora species

(Christie 1965, Hüberli et al. 2000). Successful isolation and identification of a

Phytophthora from symptomatic tissue is not sufficient to prove a disease relationship, however.

Robert Koch was a medical bacteriologist who was awarded the Nobel Prize in

Physiology and Medicine in 1905, in part for his contributions to understanding how living, parasitic organisms (pathogens) are able to cause animal diseases, such as

Anthrax and Tuberculosis (Koch 1878, Koch 1882, Pinner and Pinner 1932, Metchnikoff

1939). Koch was rigorous in his methodology. Koch’s methodological approach lead to the development of four postulates to establish an organism as the causal agent of 116 disease. One, the candidate pathogen must have a consistent association with disease symptoms. Two, the pathogen should be isolated from the diseased host and grown in pure culture. Three, the pathogen isolated in pure culture must cause the disease when it is inoculated into a healthy host under controlled laboratory conditions. Four, the pathogen must be reisolated from the inoculated host and be the same as the originally inoculated pathogen. The supporting principles of the postulates were first discussed in

Koch’s examination of wound infections (Koch 1878, Metchnikoff 1939). Examination of

Koch’s postulates is standard procedure in plant pathology (Agrios 2005), and is standard procedure for determining the pathogenicity of Phytophthora species in particular (Newhook 1959, Davidson et al. 2003, Hansen et al. 2005, Ahumada et al.

2013).

During the alder surveys in western Oregon riparian ecosystems (Chapters 2 and

3), the incidence of Phytophthora cankers was positively correlated with canopy dieback, and two species of concern were isolated from necrotic alder tissues-

Phytophthora alni subsp. uniformis Brasier & S. A. Kirk and Phytophthora siskiyouensis

Reeser & EM Hansen (Chapter 2). Eighteen additional species of Phytophthora were also identified from riparian ecosystems, although seldom from diseased alder tissues

(Chapter 3). Most numerous of these, Phytophthora taxon Oaksoil, comprised 42% of the Phytophthora isolates, prevalent in coastal streams flowing adjacent to riparian red 117

alder stands exhibiting dieback. Both P. taxon Oaksoil and P. siskiyouensis were consistently isolated in association with the diseased host displaying symptoms of canopy dieback and cankers. Phytophthora canker was correlated with canopy dieback, and isolation of P. siskiyouensis (Chapter 2). However, was canopy dieback more prevalent in alder stands that recovered P. siskiyouensis or P. taxon Oaksoil?

The primary objective of the 2010 alder survey was to search for the alder

Phytophthora in declining alder stands. The alder Phytophthora is P. alni. It causes a lethal root and collar disease of alder species in Europe. It is especially devastating in southern Britain (Gibbs et al. 1995) and it causes alder disease elsewhere in Europe. The alder Phytophthora can be recovered from locations where it is causing disease by using

baiting techniques to isolate it from soil, roots, and stream water. It can also be isolated

from bleeding bole cankers. It has not been found causing alder disease outside of

Europe. The virulent form of this destructive pathogen was not found in our survey, but

a closely related subspecies, Phytophthora alni subsp. uniformis Brasier & S.A. Kirk, was isolated rarely from diseased alder roots (Chapter 2, Aguayo et al. 2013). This subspecies was previously reported from Alaska riparian soils, but was not associated with disease

(Adams et al. 2008). Pathogenicity of Phytophthora alni subsp. uniformis from the

United States has not been tested.

118

Bole cankers (Figure 1), as well as necrotic roots, similar in appearance to those

caused by the alder Phytophthora in Europe, were regularly encountered in the riparian

survey. These symptoms were frequently accompanied by canopy dieback (Chapter 1).

However, Phytophthora siskiyouensis Reeser & EM Hansen, not P. alni, was usually

isolated (Chapter 2). Phytophthora siskiyouensis was discovered in 2003 while

monitoring areas damaged by the invasive Phytophthora ramorum Werres, De Cock &

Man in’t Veld (Reeser et al. 2007). Phytophthora siskiyouensis was initially reported to be a minor pathogen of tanoak (Notholithocarpus densiflorus (Hook. & Arn.) Manos et al.), and Oregon myrtle (Umbellularia californica (Hook. & Arn.) Nutt.; Reeser et al.

2007). Soon after description, however, it was isolated from cankers on planted black alder (Alnus glutinosa (L.) Gaertn.) in Melbourne Australia (Smith et al. 2006), and killing planted Italian alder (Alnus cordata (Lois.) Duby) in California (Rooney-Latham et al.

2007, Rooney-Latham et al. 2009). Isolates recovered from Italian alder caused cankers in artificial inoculations of alder species that occur in Oregon: red alder, and white alder

(Rooney-Latham et al. 2009), but P. siskiyouensis has not previously been reported from wild alder, and Oregon isolates of this pathogen have not been tested against alder species.

119

Figure 1) Phytophthora bole cankers on alder infected with P. siskiyouensis in western Oregon riparian ecosystems.

The most common Phytophthora species (506 identified isolates, 97% from stream water) isolated in association with alder was an informally described species P. taxon Oaksoil (Chapter 2). In 1999 a Phytophthora species was recovered from leaf debris from a healthy forest in Amance France (Hansen and Delatour 1999). It was labeled Isolate aecjv1, or P 1055, and informally described as a sterile Phytophthora species, P. taxon Oaksoil (Brasier et al 2003). It appeared to be a relatively benign 120

Phytophthora species, but very little was known about it (Hansen and Delatour 1999). It

was subsequently identified at scattered locations around the world in Poland (Belbahri

et al. unpublished in GenBank GB EU810387) and Denmark (personal communication),

including in a stream in western Oregon (Remigi et al. 2009). In 2012, a very similar, but

homothallic, Phytophthora bilorbang Aghighi & T. Burgess was described from western

Australia, killing wild blackberries (Rubus anglocandicans, Aghighi et al. 2012).

The abundance of Phytophthora taxon Oaksoil in western Oregon streams

(Chapter 2) was a concern because Phytophthora species can be very serious threats to forest health (Gibbs et al. 1995, Hansen and Delatour 1999, Goheen et al. 2002, Rizzo et al. 2002, Jung et al. 2005, Hwang et al. 2008) and investigations into the pathology of

Phytophthora species associated with alder in Oregon were only very preliminary

(Navarro 2013). In Oregon, this species appeared to be associated with stream water adjacent to diseased alder trees. It was regularly isolated from alder leaf debris as well as from the water (Figure 2). Alder leaves in streams provide one of the most accessible sources of nitrogen to fungi and microorganisms in the Pacific Northwest (Triska et al.

1975). P. taxon Oaksoil could be proliferating in streams for other reasons besides being a pathogen of alder. However, interactions in forest systems can be complex and a serious pathogen could reside in debris (Queloz et al. 2010). The prolific but potentially benign relationship between to P. taxon Oaksoil and alder needed to be examined in a 121

controlled test to confirm that isolates recovered from the Oregon population were not

virulent canker causing pathogens like the alder Phytophthora.

Figure 2) Alder leaf debris accumulates in streams.

If Phytophthora is isolated and successfully grown in a laboratory setting, then the last two of Koch’s postulates can be examined in a pathogenicity test. Pathogenicity tests are used to determine if observed field symptoms are produced under controlled conditions and caused by a single organism without the many extraneous factors that occur in a natural setting. The physical isolation of P. siskiyouensis and statistical 122

correlation of Phytophthora cankers with alder canopy dieback coupled with the very

rare recovery of P. alni subsp. uniformis supported the hypothesis that Phytophthora disease in western Oregon was caused by Phytophthora siskiyouensis and not P. alni; but isolation, and association with disease, without knowing whether P. siskiyouensis was able to cause cankers was not enough. Pathogenicity testing needed to be completed.

Evidence suggested that in the United States, in western Oregon in particular, P. siskiyouensis was the causal agent of Phytophthora cankers. However, two other

Phytophthora species that were recovered were also a concern: P. alni subsp. uniformis and P. taxon Oaksoil.

We hypothesized that P. siskiyouensis was the causal agent of Phytophthora cankers on alder in western Oregon riparian ecosystems. The goal was to complete

Koch’s postulates. The major objective was to examine the pathogenicity of P. siskiyouensis, P. alni subspecies uniformis and P. taxon Oaksoil to alder species from western Oregon.

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The goal of this chapter was to complete Koch’s postulates for three species of

Phytophthora associated with declining alder trees in western Oregon riparian ecosystems. To this end, we compiled and expanded on survey reports (Chapter 2 and

Chapter 3) for the distribution of the species and their association with canopy dieback.

We tested their pathogenicity on alder seedlings, and reisolated the pathogens, thus completing Koch’s postulates.

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MATERIALS AND METHODS Survey efforts - Three surveys were conducted in western Oregon in association with alder. Survey efforts and Phytophthora sampling and processing is briefly described below. Detailed survey efforts and detailed Phytophthora species sampling and processing methods are described elsewhere (Chapter 1, Chapter 2 and Chapter 4). A list of recovered isolates will be provided.

The first survey was conducted on 88 transects established across western

Oregon. This survey focused on alder symptoms (Chapter 2) and was a general assessment of Phytophthora associated with alder tree species across western Oregon

(Chapter 3). To assure reasonably uniform sampling across all of western Oregon, transects were laid out in riparian alder stands in three subregions: Coastal streams draining into the Pacific Ocean, Willamette Valley streams draining into the Willamette

River, and Southern streams draining into the Umpqua River and rivers south of that. In most cases, three transects were established along a stream, in the upper watershed with minimal human disturbance, in the mid watershed, often with agricultural lands nearby, and in the lower watershed, often in the midst of rural residential development and in more developed areas at the urban wildland interface. The Phytophthora based aspects of this survey focused on recovery from soil baits and root baits (the rhizosphere), and stream water. Bark samples were occasionally but not systematically 125 taken from this initial survey effort. The second survey was from a subset of 18 coastal transects. These transects were targeted because of bleeding canker symptoms. The second survey focused on recovery from bleeding cankers and surface sterilized diseased roots. In addition, in 2012 we reisolated from Phytophthora cankers from a transect in the Willamette Valley because cankers were initially culture negative. The third survey was from two transects in the coastal area of western Oregon. These sites were targeted to examine all Phytophthora species, and the seasonality of P. taxon

Oaksoil associated with streams and alder leaf debris.

Sampling efforts - Alder trees were sampled for Phytophthora species.

Phytophthora species were examined in the area of diseased trees and from diseased tissue directly. Samples were from bark cankers, from soil baits, root baits, surface sterilized roots, from leaf material in streams, leaves on trees and stream water beneath alder canopies. Bark pieces were cultured onto oomycete selective media (CARP: cornmeal agar, 10 ppm rifamycin SV sodium salt, 20 ppm Delvocid [50% natamycin salt],

200 ppm ampicillin sodium salt, Reeser et al. 2011). Root samples were washed to remove soil, soaked and cleaned with a solution of sodium hypochlorite. Clean root pieces were examined for cankers, necrotic spots, and/or water soaked lesions.

Symptomatic pieces of roots were directly plated onto selective H/2 media: cornmeal agar, 10 ppm rifamycin SV sodium salt, 20 ppm Delvocid [50% natamycin salt], 200 ppm 126 ampicillin sodium salt, and 25 ppm hymexazol (Reeser et a. 2011). For baiting soil and root samples were collected, brought to the laboratory, flooded with deionized water; and baited with mature Rhododendron leaves. Bait pieces remained in place for 72 h; and then bait pieces were removed, cleaned and partially submerged into H/2 media.

For water filtration stream water was collected and filtered. Filters were inverted and placed onto VARP+ media: 15 g Bactoagar, 50 mL V8 stock, 10 ppm rifamycin SV sodium salt, 20 ppm Delvocid [50% natamycin salt], 200 ppm ampicillin sodium salt, 30 ppm

Benlate [benomyl 50WP], and 50 ppm hymexazol (Chapter 2). Filters were incubated on media surface for a 72 h at 20 °C. Phytophthora colonies were sampled and sub-cultured onto CARP media: cornmeal agar, 10 ppm rifamycin SV sodium salt, 20 ppm Delvocid

[50% natamycin salt], 200 ppm ampicillin sodium salt (Reeser et al. 2011, Chapter 1).

Alder leaf debris samples were collected in streams beneath and adjacent to alder tree canopies. Leaves were also collected from downed trees and branches with submerged leaves in stream water. From one site attached alder leaves were collected.

Unidentifiable leaves were not processed. Hole punched leaf pieces were gently submerged into VARP+.

Phytophthora species determination - Phytophthora were characterized morphologically to genus on culture plates. The morphological characters used to distinguish Phytophthora were rate of growth, and branching pattern. Often other 127

morphological characters, such as sporangia, oogonia and chlamydospores were also

present. To determine if an ambiguous isolate was Phytophthora it was grown in water

culture to examine zoosporogenesis. Phytophthora species isolates were DNA extracted.

After extraction DNA was PCR amplified and sequenced in the Cytochrome c oxidase

(Cox) spacer region (Martin & Tooley 2003) of the mitochondrial DNA. Sequences were analyzed manually and aligned using ClustalX 2.0.10 software. Species determination was based on a match to a known reference isolate. If an isolate did not match a reference isolate then DNA was PCR amplified and sequenced in the internal transcribed spacer region (ITS). Species determination was based on a match to a known reference isolate in the ITS region.

Fishers exact test for count data was performed to examine the relationship between canopy dieback within a transect and successful isolation of two Phytophthora species: P. siskiyouensis and P. taxon Oaksoil from a transect. The number of alder trees, within a transect, with and without canopy dieback and P. siskiyouensis were compared to alder trees with and without canopy dieback and P. taxon Oaksoil but not P. siskiyouensis (and with no P. siskiyouensis recovered from a transect located upstream along the same watershed). Data for canopy dieback was from the 2010 survey.

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Alder for the inoculation trial - Alder seedlings from different lots were used in a pathogenicity experiment. Red alder (Alnus rubra) 1.5 m tall containerized seedlings were purchased from Seven Oaks Native Nursery in Albany, Oregon in February 2011. A set of one-year-old bare root seedlings were purchased from Seven Oaks Native Nursery in January 2012. White alder (Alnus rhombifolia) 2 m tall containerized seedlings were also purchased from Seven Oaks Native Nursery in January 2012. All alder were from seed sources in western Oregon. The one-year-old bare root seedlings were planted in

650 mL tubes with potting mix. The containerized seedlings from 2011 and 2012 were transferred to larger pots in 2012. A sample of the potting mix used for transplanting was baited and baits were plated on oomycete selective media, CARP (corn meal agar amended with 10 ppm natamycin, 200 ppm Na-ampicillin, 10 ppm rifamycin-SV), to test for presence of Phytophthora or Pythium species. Each seedling was tagged for identification. The seedlings were grown outdoors at Oregon State University until June

2012 and were watered by rainfall during the rainy season and with a sprinkler system twice daily during the dry season.

Stem inoculation trial - Every alder, regardless of lot, species, or size represented a unique alder unit. Two weeks prior to the pathogenicity test alder units were moved into a greenhouse. A stem inoculation pathogenicity test was conducted in July of 2012 and lasted 15 days. The greenhouse temperature averaged 22 °C. Two isolates of each 129

of the three Phytophthora species (P. siskiyouensis, P. alni subsp. uniformis, and P. taxon

Oaksoil) were used. Eight alder units were inoculated with P. siskiyouensis (4 alder units for each isolate), eight alder units were inoculated with P. alni subsp. uniformis (4 alder units for each isolate), six alder units were inoculated with P. taxon Oaksoil (3 alder units each isolate) and five alder units were inoculated with an uncolonized CA plug. Which alder unit received a particular inoculation treatment was by random assignment.

Randomization was with a random number generator using seedling tag numbers.

A 2 cm flap was cut into each containerized alder seedling, transversely along the stem about 25cm above the soil line. Each alder seedling in a tube was pricked with a pin about 15 cm above the soil line. One inoculation was applied to each alder unit by either inserting a colonized 6 mm CA agar plug into the cut or laying a colonized 6 mm

CA agar plug over the pin prick wound. Sterile technique was used, flaming tools between each inoculation event. The inoculated area was then wrapped in dry cheesecloth followed by parafilm. Stems were watered with tap water after inoculation to dampen the inoculated area (cheesecloth extended slightly past the parafilm and the water wicked in). The inoculation area was covered with aluminum foil. Alder units were haphazardly regrouped so that trees with the same treatment were not clustered together. Then each alder unit was placed into a water container to keep the root area 130 wet. After a 72 h period the water containers were removed and alder units were watered daily.

Following the 15 day incubation period, foil, parafilm, and cheesecloth were removed. The alder units were visually assessed for canker symptoms. The outer bark at the leading edge of the canker was scraped with a scalpel to determine the true outer edge of the canker margin. If no obvious canker was visible the whole inoculation area was lightly scraped to remove the outer bark and the darkened tissue area was measured prior to visible oxidation. To prevent cross contamination the scalpel was flame sterilized after scraping each alder unit. The canker length was measured to the nearest mm as the distance between the two farthest canker endpoints.

To confirm symptoms were the results of the inoculated pathogen and not any other external or internal factor, the pathogen was reisolated following incubation. Bark pieces from the leading edge of the canker margin were removed. Bark pieces were carefully submerged into VARP+ media. Plates were incubated for at least 10 days. Each plate was examined for morphological features characteristic of the Phytophthora species the alder unit was inoculated with.

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Modeling a treatment effect – To test the hypothesis that P. siskiyouensis was

the causal agent of Phytophthora cankers on alder trees in Western Oregon, and to

examine the pathogenicity of Phytophthora siskiyouensis, P. alni subspecies uniformis and P. taxon Oaksoil the model below was constructed to describe the response. The response was the canker length that develops on the alder host following a 15 day incubation period of the pathogen on the host. The model takes into account two potential errors: 1) That isolates of the same Phytophthora species, although grouped

together as a species (nested), may behave differently (because we know that isolates

of the same species can behave differently), and 2) that each individual alder

unit/pathogen combination may have unique attributes leading to a different response

because each alder unit/pathogen combination is unique (each tree is a unique

biological unit).

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Yijk= α0 + α1ind.siski + α2ind.alnii + α3ind.oaki + bj + εijk

where:

Yijk is the canker length from the ith inoculation treatment from the jth isolate on the kth alder tree, i=1, 2, 3, 4, j=1, 2,…7, k=1, 2…27

α0 is the mean canker length on the alder unit inoculated with the negative control treatment,

α1 is the difference from the negative control treatment in the mean canker length from the P. siskiyouensis inoculation treatment,

α2 is the difference from the negative control treatment in the mean canker length from the P. alni inoculation treatment,

α3 is the difference from the negative control treatment in the mean canker length from the P. taxon Oaksoil inoculation treatment, ind.siski is 1 when the ith treatment is P. siskiyouensis inoculation treatment and 0 otherwise, ind.alnii is 1 when the ith treatment is P. alni inoculation treatment and 0 otherwise,

ind.oaki is 1 when the ith treatment is P. taxon Oaksoil inoculation treatment and 0 otherwise,

2 bj is the random effect of isolate on mean canker length, bj ~ N(0, σb ) and

Cov(bj,bj’)=0

εijk is the random effect associated with each alder unit pathogen 2 combination on mean canker length, εijk ~ N(0, σ ), and Cov(εijk,εi’j’k’)=0, and bj, εijk are all independent.

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The natural logarithmic scale was applied to the response based on a graphical

assessment of plots. A natural log transformation of canker length was found to stabilize

the variance, and the residuals appeared to be adequately symmetric and

approximately normal after the transformation (Figure 3).

(a) (b)

Normal Q-Q Plot of Residuals w/o Log transformation Normal Q-Q Plot of Residuals w/ Log transformation 2 0.6 0.4 1 0.2 Sample Quantiles Sample Quantiles Sample Quantiles Sample Quantiles 0 0.0 -0.2 -1 -1 -0.4 -2 -2 -0.6

-2 -1 0 1 2 -2 -1 0 1 2

Theoretical Quantiles Theoretical Quantiles

Figure 3) Normal quantile-quantile plot of residuals from the model before (a) and after (b) applying a natural log transformation to canker length.

The model was constructed to determine (1) if there was evidence for a treatment effect and, (2) examine if the canker length was estimated to be longer than negative controls for P. siskiyouensis, P. alni subsp. uniformis, and P. taxon Oaksoil. The response was transformed (natural log). The ratio (treatment/control) of the median canker length was used in place of the difference from the negative control treatment in mean canker length from each inoculation treatment. The median was used in place of 134 the mean because it is the correct measurement following a log transformation (Ramsey and Schafer 2002).

135

RESULTS

In association with alder stands in western Oregon, a lot of Phytophthora was recovered. A total of 1190, identified Phytophthora isolates were collected from the 88 transects in the initial survey. Phytophthora species were isolated from 83 (94%) transects. Four isolates from two transects were identified as Phytophthora alni spp. uniformis (Table 1) from the survey focusing on diseased alder roots. All came from necrotic lesions on red alder roots. Phytophthora siskiyouensis was isolated 74 times, mainly from bole cankers and diseased roots on red and white alder, and from water and alder leaf debris floating in the streams (Table 1). P. siskiyouensis was recovered from 17 transects. P. taxon Oaksoil was isolated 506 times from 58 transects; almost all

(97%) isolates were from water and leaf debris. Only the P. taxon Oaksoil isolates used in pathogenicity testing are included in Table 1.

The complete list of Phytophthora siskiyouensis and Phytophthora alni spp. uniformis isolates recovered from bark cankers, soil baits, root baits, surface sterilized roots, and stream water is included in Table 1.

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Table 1. P. siskiyouensis, and P alni subsp. uniformis isolated in pure culture and recovered from diseased alder tissue. Isolates included were also recovered in association with alder stand with canopy dieback in western Oregon riparian ecosystems and include the two isolates of P. taxon Oaksoil used in pathogenicity testing.

Isolate Species County isolated from 118-R-1K.1† alni subsp. uniformis Lane red alder root 118-R-1J.1 alni subsp. uniformis Lane red alder root 118-R-1101711.4 alni subsp. uniformis Lane red alder root 110-R-1N.1† alni subsp. uniformis Douglas red alder root 118-W-2K.20† taxon Oaksoil Lane Stream water 114-W-1.6† taxon Oaksoil Lane Stream water 117-B-4S.1 † siskiyouensis Lane red alder bark canker 117-R-2S.3 † siskiyouensis Lane red alder root 108-G-1G.2 siskiyouensis Tillamook red alder leaf debris 108-G-1G.4 siskiyouensis Tillamook red alder leaf debris 108-G-2G.13 siskiyouensis Tillamook red alder leaf debris 108-G-2G.8 siskiyouensis Tillamook red alder leaf debris 117-N-1S.2 siskiyouensis Lane red alder leaf debris 117-N-1S.3 siskiyouensis Lane red alder leaf debris 118-G-1R.1 siskiyouensis Lane red alder leaf debris 118-GSLD-3.1 siskiyouensis Lane red alder leaf debris 118-GSLD-3.3 siskiyouensis Lane red alder leaf debris 118-GSLD-3.4 siskiyouensis Lane red alder leaf debris 118-GSLD-3.5 siskiyouensis Lane red alder leaf debris 118-GSLD-3.7 siskiyouensis Lane red alder leaf debris 121-G-3H.1 siskiyouensis Lincoln red alder leaf debris 118-N-G.6 siskiyouensis Lane red alder leaf debris 110-B-4N.1 siskiyouensis Lincoln red alder bark canker 121-B-1J.1 siskiyouensis Lincoln red alder bark canker 121-B-1J.2 siskiyouensis Lincoln red alder bark canker 121-B-1J.3 siskiyouensis Lincoln red alder bark canker 121-B-2J.1 siskiyouensis Lincoln red alder bark canker 222-29-B.1 siskiyouensis Lane red alder bark canker ST33BLRH1.1 siskiyouensis Lane white alder bark canker ST33BLRH1.2 siskiyouensis Lane white alder bark canker ST33BLRH1.3 siskiyouensis Lane white alder bark canker ST33BLRH1.4 siskiyouensis Lane white alder bark canker ST33BLRH2.1 siskiyouensis Lane white alder bark canker ST33BLRH2.2 siskiyouensis Lane white alder bark canker ST33BLRH3.1 siskiyouensis Lane white alder bark canker 118-DL-1H.2 siskiyouensis Lane red alder leaf at stream surface 117-4-R.1 siskiyouensis Lane red alder root bait 137

201-36-R.1 siskiyouensis Jackson white alder root bait 201-36-R.2 siskiyouensis Jackson white alder root bait 103-9-S.1 siskiyouensis Tillamook red alder soil bait 104-W-1.1 siskiyouensis Lincoln stream water 107-W-2.12 siskiyouensis Tillamook stream water 109-W-2.1 siskiyouensis Tillamook stream water 113-W-1.2 siskiyouensis Lane stream water 113-W-2.10 siskiyouensis Lane stream water 113-W-2.11 siskiyouensis Lane stream water 118-W-1H.1 siskiyouensis Lane stream water 118-W-1R.5 siskiyouensis Lane stream water 119-W-2.12 siskiyouensis Lincoln stream water 119-W-2.3 siskiyouensis Lincoln stream water 119-W-2.5 siskiyouensis Lincoln stream water 121-W-1G.3 siskiyouensis Lincoln stream water 121-W-1H.2 siskiyouensis Lincoln stream water 121-W-1R.2 siskiyouensis Lincoln stream water 121-W-1R.3 siskiyouensis Lincoln stream water 121-W-2I.9 siskiyouensis Lincoln stream water 121-W-2J.6 siskiyouensis Lincoln stream water 126-W-2.9 siskiyouensis Tillamook stream water 108-R-2G.1 siskiyouensis Tillamook red alder root 114-R-3N.4 siskiyouensis Lane red alder root 117-R-2L.2 siskiyouensis Lane red alder root 117-R-2L.3 siskiyouensis Lane red alder root 117-R-2S.2 siskiyouensis Lane red alder root 117-R-2S.4 siskiyouensis Lane red alder root 117-R-3S.5 siskiyouensis Lane red alder root 117-R-5S.2 siskiyouensis Lane red alder root 118-R-G.2 siskiyouensis Lane red alder root 118-R-G.3 siskiyouensis Lane red alder root 118-R-G.4 siskiyouensis Lane red alder root 118-R-G.6 siskiyouensis Lane red alder root 118-R-1H.11 siskiyouensis Lane red alder root 118-R-1H.13 siskiyouensis Lane red alder root 118-R-1H.14 siskiyouensis Lane red alder root 118-R-1H.15 siskiyouensis Lane red alder root 118-R-1H.17 siskiyouensis Lane red alder root 118-R-1H.18 siskiyouensis Lane red alder root 118-R-1H.5 siskiyouensis Lane red alder root 118-R-1H.6 siskiyouensis Lane red alder root 118-R-1H.7 siskiyouensis Lane red alder root 118-R-1H.8 siskiyouensis Lane red alder root

†Phytophthora species isolates used in the pathogenicity testing. 138

Correlating canopy dieback with Phytophthora species – Canopy dieback was more prevalent in riparian alder trees from transects with P. siskiyouensis than from transects with P. taxon Oaksoil but without P. siskiyouensis (70% and 35%, respectively)

(Table 2). There was strong evidence the number of alder trees with canopy dieback was not the same from transects with P. siskiyouensis compared to transects without P. siskiyouensis but with P. taxon Oaksoil (p-value < 0.0001). From Fishers exact test the odds of canopy dieback for alder trees were 4.2 times greater (95% CI: 3.2, 5.5) from transects with P. siskiyouensis compared to transects with P. taxon Oaksoil but not P. siskiyouensis (and no P. siskiyouensis from upstream transects). From transects with only P. siskiyouensis 81% had canopy dieback. For most (88%) P. siskiyouensis positive transects, more than 50% of alder trees had canopy dieback (Table 2). For most transects (61%) the opposite was true (less than 50% of alder trees had canopy dieback) if P. taxon Oaksoil was present but not P. siskiyouensis (Table 2). 139

Table 2) Comparisons of alder canopy dieback from transects with P. siskiyouensis vs P . taxon Oaksoil Phytophthora siskiyouensis locations Location of transect number of alder trees presence of N° W° with canopy dieback without canopy dieback Phytophthora alni * 44.7855 123.7867 28 17 45.3229 123.7680 11 2 45.3140 123.7815 18 1 45.3131 123.8347 8 4 43.8152 123.6519 19 7 + 44.1536 123.9505 16 10 44.1004 123.9372 18 5 44.1331 124.0867 9 1 44.1301 124.0937 7 3 44.1332 124.1204 16 5 + 44.2775 123.9621 8 6 44.3069 124.0744 24 5 45.4328 123.7271 21 0 42.0312 123.0895 35 6 43.6739 122.7303 31 12 44.0459 123.0271 3 28 45.1340 123.8949 4 8 total alder trees 276 120 Phytophthora taxon Oaksoil locations without positive isolation of Phytophthora siskiyouensis Location of transect number of alder trees presence of N° W° with canopy dieback without canopy dieback Phytophthora alni * 44.6775 123.2613 6 16 44.6060 123.0779 7 4 44.7123 122.6095 4 18 44.7119 122.7181 6 4 45.2239 123.4818 9 3 45.1334 123.4899 8 3 45.0780 123.4869 3 13 44.8720 123.4700 6 4 44.8656 123.4351 2 22 45.3913 122.4934 8 11 44.9692 122.6401 2 11 44.9992 122.6664 4 4 45.0350 122.7588 10 17 43.9579 122.8164 8 0 43.5942 123.0341 8 3 44.0134 122.9839 6 12 45.0851 123.7911 1 24 45.1231 123.8799 5 17 44.7880 123.8157 9 13 44.7307 123.8342 10 8 42.7171 124.3819 7 1 42.7385 124.4067 17 5 42.7748 124.4712 16 10 45.4589 123.5907 4 21 44.3439 123.6857 13 1 44.3989 123.8520 15 4 ? 42.7933 122.4671 17 20 42.0659 123.1091 6 34 42.1804 123.3522 1 84 42.9016 122.9312 8 29 43.0367 122.8089 5 23 43.0348 123.7405 8 37 42.1464 123.4350 10 56 42.0451 123.7472 27 18 43.3983 122.9265 45 31 43.2392 122.9339 13 14 43.7070 122.7438 7 10 42.0353 124.1083 11 43 total alder trees 352 648 * subsp. uniformis 140

Pathogenicity test results from the model- The alder trees inoculated with P. siskiyouensis or P. alni subsp. uniformis had large cankers when compared to cankers from P. taxon Oaksoil inoculated trees or cankers from the inoculation process (Figure

4). There was strong statistical evidence for a pathogen treatment effect based on the f statistic (F3,3 = 211.7, p-value = 0.0005 ). Based on the p-value from the t-statistic, the

ratio of median canker length was estimated to be longer than negative controls for

both P. siskiyouensis (p-value = 0.0004), and P. alni subsp. uniformis (p-value = 0.0003),

but not for P. taxon Oaksoil (p-value = 0.1920). Since a natural log transformation was

applied to the response variable, the ratio (pathogen/control) of the medians is

reported in place of the difference of the means (Table 3).

Table 3.) Ratio (pathogen/control) of the medians estimate for canker length caused by the pathogen. The 95% confidence intervals (CI) based on estimates. Comparison is with n= 5 control treatments.

Pathogen Treatment n Ratio of Medians 95% CI Phytophthora taxon Oaksoil 6 1.4 0.7 , 2.9 Phytophthora siskiyouensis 8 39.9 20.8 , 76.8 Phytophthora alni 8 45.2 23.5 , 87.0

To confirm the observed symptoms were the results of the inoculated pathogen

and not another external or internal factor, the pathogen was reisolated from bark

tissue onto Phytophthora selective media following incubation. The appropriate

Phytophthora species was successfully reisolated from all inoculated alder trees. The 141 reisolated pathogens were recognizable to species based on morphology. P. alni subsp. uniformis produced mostly large (40 - 45 u) but variably sized oogonia with some bullate protuberances. Antheridia were amphigynous, mainly 2 celled, but some antheridia were single celled. P. siskiyouensis exhibited globose small (25 - 30 u diameter) oogonia with paragynous antherida. P. taxon Oaksoil hyphae grew in a characteristic straggly colony formation, with occasional sporangia but no oogonia. Negative control treatment trees did not yield Phytophthora species on reisolation.

Figure 4. From left to right cankers resulting from inoculation with: a carrot agar plug, P. taxon Oaksoil, P. siskiyouensis, and P. alni subsp. uniformis. Each alder tree unit pictured was stem wound inoculated over a pinprick wound. Trees were incubated in the greenhouse for 15 days prior to examining cankers.

142

DISCUSSION

Dieback of riparian alder trees in western Oregon is a continuing concern. In

Chapter 2 it was shown that dieback may have different causes in different trees, but

that the strongest correlation in western Oregon riparian ecosystems is with

Phytophthora bleeding cankers on tree boles. Trees with Phytophthora cankers were more likely to exhibit dieback than trees without cankers and P. siskiyouensis was isolated from bole cankers. In Chapter 3 it was shown that P. siskiyouensis was isolated

from all substrates around diseased alder trees. In this chapter we established the

statistical association between presence of P. siskiyouensis and increased dieback

compared to the relatively benign P. taxon Oaksoil, and demonstrated the pathogenicity

of P. siskiyouensis to alder, completing Koch’s Postulates. Koch’s postulates were

completed for P. siskiyouensis; strong evidence P. siskiyouensis was the causal agent of

the Phytophthora cankers observed on alder trees in western Oregon USA.

Phytophthora canker of alder is a bole canker caused by Phytophthora. It has

only been found to be caused by Phytophthora siskiyouensis in western Oregon.

Phytophthora canker of alder is characterized by bleeding cankers on both red alder

(Alnus rubra) and white alder (Alnus rhombifolia). Bleeding cankers are sometimes

accompanied by cracked outer bark. Cankered inner bark tissue is delimited by a dark 143 boundary. Trees with Phytophthora canker of alder are likely to have canopy dieback and Phytophthora infected roots.

Figure 5) Symptoms produced by P. siskiyouensis in the field

(left) were similar to symptoms observed in the inoculation trial (right). Symptoms in the field were more dramatic, in this instance, because they were on larger host trees and developed over a longer time.

Symptoms of Phytophthora canker in the field in Oregon and on inoculated sapling and small trees were similar to those caused by the alder Phytophthora P. alni subsp. alni, in Europe. P. alni subsp. alni was not found in Oregon, however, and P. alni 144

subsp. uniformis was uncommon in western Oregon (Table 2). P. alni subsp. uniformis was recovered from necrotic alder roots (Chapter 2) of diseased alder trees, four times

(Table 1). P. alni is not considered to be the causal agent of Phytophthora cankers in western Oregon at this time P. siskiyouensis is. Isolation of P. alni subsp. uniformis did not match the widespread pattern of the canker disease or alder canopy dieback but for

P. siskiyouensis the pattern matched. Even though results from the pathogenicity test suggest P. alni subsp. uniformis does have the potential to cause cankers the cankers caused by P. siskiyouensis were as severe; and the presence of P. siskiyouensis correlates with the pattern of disease. It was not surprising that P. alni subsp. uniformis

was able to make large cankers, because this subspecies is one of three responsible for a

lethal root and collar disease of alder in Europe. Before these survey efforts P. alni subsp. uniformis had not been recovered from Oregon or from anywhere in the United

States in association with red alder trees, and its pathogenicity to alder trees that occur in western Oregon, had not been examined. P. alni subsp. alni, is the most virulent subspecies on alder in Europe, this subspecies, to this date, has not been recovered from the United States and was not recovered here.

145

Figure 6) Alder canker lengths in the pathogenicity test- Black circles represent the ratio of the medians of canker length adjoined to 95% CI error bars. Red hash line represents no difference from the negative control response. Mean canker length for the negative control was 0.1 cm. Red diamonds represent mean canker length after a 15 day incubation period. The gray area suggests an area of canker lengths resulting from a weak pathogen.

P. taxon Oaksoil was also recovered in association with alder trees in western

Oregon (Table 2). However, most isolates were recovered from streams and it was

seldom recovered from the rhizosphere around roots or in the soil (Chapter 3). Alder

stands with P. taxon Oaksoil had a much smaller proportion of alder trees with canopy

dieback than alder stands that also had P. siskiyouensis. In the pathogenicity test, the

cankers resulting from P. taxon Oaksoil were small and did not differ significantly from 146 the negative control treatment (Figure 6). The cumulative evidence from this study suggests that P. taxon Oaksoil is a weak pathogen, at best, of alder trees in western

Oregon.

Further research should examine unexplored aspects of Phytophthora canker of alder and the relationship between Phytophthora species and alder trees. All trees we observed were in riparian areas, it is uncertain if trees outside of riparian areas occur in conditions conducive to the development of Phytophthora canker. Further monitoring could check for increased incidence of P. alni subsp. uniformis, P. siskiyouensis or

Phytophthora canker disease using baseline data from these survey efforts. In addition, the pathogenicity of P. taxon Oaksoil on alder foliage could be examined.

Other organisms in forests can cause bleeding symptoms on alder tree boles.

Boring beetles, or the fungi they transmit, cause small bleeding canker spots on the boles of trees, but were accompanied by bore holes and Phytophthora was not isolated from these cankers. The dead tissue just barely extended past the bore hole area (Figure

7 center). Occasionally trees with Phytophthora cankers also had boring insects, but this was not consistent across the landscape. Cankers that were similar in appearance to

Neonectria canker (Cootsona 2006, type 1 canker) had a tarry bleeding spot on the outer bark similar to some Phytophthora canker symptoms, but the inner bark canker of 147

Neonectria (Figure 7 right) in this study was not delimited by a distinctive dark outer boundary, characteristic of Phytophthora canker of alder (Figure 7 left), although a faint boundary was somewhat evident (Figure 7 right arrow). Trees affected by Neonectria type canker were usually found in the shaded understory and did not recover

Phytophthora; alder stands were not seriously affected in Washington (Cootsona 2006) and do not appear to be seriously affected in Oregon. Phytophthora cankers appeared different from other types of cankers, and often had stacked cankers with multiple dark boundaries (Figure 7 left arrows). Trees with Phytophthora canker had ready access to light, but were in decline.

Figure 7) The inner bark of bleeding cankers caused by P. siskiyouensis on alder looked very different from cankers associated with beetles or from Neonectria. From left to right – Phytophthora canker stacked with multiple dark boundaries (arrows), bore holes cankers from insects, and Neonectria canker with a faint boundary (arrow).

148

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Cootsona, C.2006. Identification and Distribution of Neonectria Major Causing Cankers on Red Alder (Alnus Rubra), MS thesis, College of Forest Resources University of Washington, Seattle WA.

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Metchnikoff E. 1939. The Founders of Modern Medicine Pasteur Koch Lister. Walden Publications. New York. Navarro S. 2013. Pathogenicity of Phytophthora species from Oregon waterways. M.S. thesis, Department of Botany and Plant Pathology Oregon State University, Corvallis, Oregon U.S.A. Newhook FJ. 1959. The association of Phytophthora spp. with mortality of Pinus radiata and other conifers. New Zealand Journal of Agricultural Research 2(4): 808-843. doi: 10.1080/0028233.1959.10422840 Pinner and Pinner .1932. The Aetiology of Tuberculosis. English translation. National Tuberculosis Foundation. New York USA. Queloz V, Grünig CR, Berndt R, Kowalski T, Sieber TN, and Holdenrieder O. 2010. Cryptic speciation in Hymenoscyphus albidus. Forest Pathology 41: 133–142. doi:10.1111/j.1439-0329.2010.00645. Ramsey FL, and Schafer DW. 2002. The Statistical Sleuth. Second Edition. Wadsworth Group. USA. p71-73. Remigi P, Sutton W, Reeser PW, and Hansen EM. 2009. Characterizing the community of Phytophthora species in an Oregon forest stream. In Goheen EM, Frankel SJ, editors. (tech. coords.), Proceedings of the Fourth Meeting of the International Union of Forest Research Organizations (IUFRO) Working Party S07.02.09: Phytophthoras in Forests and Natural Ecosystems. Gen. Tech. Rep. PSW-GTR 221: 311–314 U.S. Department of Agriculture, Forest Service, Pacific Southwest Research Station, Albany, CA, USA. Reeser PW, Hansen EM, and Sutton W. 2007. Phytophthora siskiyouensis, a new species from soil, water, myrtlewood (Umbellularia californica) and tanoak (Lithocarpus densiflorus) in southwestern Oregon. Mycologia 99: 639–643. Rizzo DM, Garbelotto M, Davidson JM, Slaughter GW, and Koike ST. 2002. Phytophthora ramorum as the cause of extensive mortality of Quercus spp. and Lithocarpus densiflorus in California. Plant Disease. 86(3): 205-214. Rooney-Latham S, Blomquist CL, Pastalka T, and Costello L. 2007. First Report of Phytophthora siskiyouensis causing disease on Italian alder in Foster City, CA, USA. Phytopathology 97: S101. 151

Rooney-Latham S, Blomquist CL, Pastalka T, Costello L. 2009. Collar rot on Italian alder trees in California caused by Phytophthora siskiyouensis. Plant Health Progress. doi: 10.1094/PHP-2009-0413-01-RS. Smith IW, Cunnington J, Pascoe I. 2006. Another new? Species of Phytophthora on alder ‘down under’ (Australia). In Third Workshop of IUFRO Working Party 7.02.09 Phytophthora in Forests and Natural Ecosystems. Edited by Braiser C, Jung T, Oßwald WF. Farnham, UK: Forest Research. Thines M. 2013. Taxonomy and phylogeny of Phytophthora and related Oomycetes. In Phytophthora a Global Perspective. Editor: Lamour K. University of Tennessee. Knoxville, USA. CABI plant protection series; 2. pp. 11-18. Triska FJ, Sedell JR, Buckley B. 1975. The processing of conifer and hardwood leaves in two coniferous forest streams: II. Biochemical and nutrient changes. Verh. Internat. Verein. Liminol. 19: 1628-1639. 152

Chapter 5. Phytophthora obrutafolium species nov., an Alnus rubra leaf saprotroph closely related to P. bilorbang and P. taxon Oaksoil ss

INTRODUCTION

Five hundred and six isolates of the informally described sterile species

Phytophthora taxon Oaksoil were recovered in the examination of Phytophthora de Bary

associated with red alder (Alnus rubra Bongard) in western Oregon riparian ecosystems

(Chapter 3). For the first time Phytophthora siskiyouensis Reeser & E. M. Hansen was

recovered from bark cankers of alder trees in natural ecosystems (Chapter 2) and is

described as the canker causing pathogen of red alder and white alder (Alnus

rhombifolia) in Oregon USA (Chapter 4). Twenty Phytophthora species were recovered

in total including the alder Phytophthora, Phytophthora alni spp. uniformis Brasier & S.

A. Kirk (Chapter 2). However, apparently abundant in association with riparian

ecosystems, 43% of the 1190 isolates recovered were P. taxon Oaksoil. In this chapter P.

taxon Oaksoil from western Oregon is described as new sterile species Phytophthora

obrutafolium sp. nov. Then the ecological role of Phytophthora obrutafolium sp. nov. is examined.

Species in the genus Phytophthora are eukaryotic organisms, classified within the diploid Oomycetes in the Stramenopiles (Cavalier-Smith 1986, Baldauf et al. 2008).

The genus contains more than 100 species (Hansen et al. 2012, Kroon et al. 2012) that 153 occupy an assortment of aquatic and terrestrial habitats (Erwin and Ribeiro 1996).

Phytophthora can be grouped into branches (clades) of a relatedness tree using the nuclear internal transcribed spacer region (ITS) to classify and understand these organisms in an evolutionary context (White et al. 1990, Cooke et al. 2000). Each clade has life history attributes common to the clade (Kroon et al 2012). Clades are numbered

1 through 10. Sterility is a defining characteristic of many species within clade 6 (Brasier et al. 2003). Twelve of twenty-three recognized species within the clade are sterile and only three species outside of clade 6 are sterile with no more than one sterile species in any other clade (Kroon et al. 2012). Clade 6 Phytophthora species in general tend to be associated with forest streams. Phytophthora gonapodyides (H.E. Petersen) Buisman, for example, has a cosmopolitan distribution and is documented from stream surveys around the world including Oregon stream surveys (Sutton et al. 2009, Hwang et al.

2009, Reeser et al. 2011, Jung et al. 2011, Chapter 2). Clade 6 species may be associated with stream water and forest debris without necessarily being associated with disease

(Hansen and Delatour 1999, Hansen et al. 2012, Chapter 2).

In 1999 a clade 6 Phytophthora isolate known as P 1055 or aecjv1, was recovered from oak leaf debris in an oak forest in France (Hansen and Delatour 1999). This isolate was later informally described as the sterile species P. taxon Oaksoil (Brasier et al 2003).

In 2012, Phytophthora bilorbang Aghighi & T. Burgess, a homothallic species, was 154

described that matched isolate aecjv1 in the nuclear internal transcribed spacer (ITS)

region. This species was described as the pathogen responsible for the decline of Rubus

anglicandicans in Western Australia (Aghighi et al. 2012).

P. taxon Oaksoil sensu lato from Oregon differs from P. bilorbang in western

Australia and P. taxon Oaksoil sensu stricto (isolate aecjv1) from France, although it too also matched in the ITS region. Each is associated with different hosts, P. bilorbang with

Rubus anglicandicans (Aghighi et al. 2012) and P. taxon Oaksoil ss with Quercus robur

(Hansen and Delatour 1999). P. taxon Oaksoil sl from Oregon on the other hand,

appeared to be associated with and prolific around red alder (Alnus rubra). Oregon

isolates also differed genetically by a SNP (consistently) from both P. bilorbang and P. taxon Oaksoil ss, in the mitochondrially encoded spacer region between the cytochrome oxidase subunit genes 1 and 2. These differences plus P. bilorbang was sexually homothallic while both P. taxon Oaksoil ss and P. taxon Oaksoil sl from Oregon were sexually sterile, suggested that although these three were closely related, each was a unique species, differing in host association, geography, haplotype, and for P. bilorbang and P. taxon Oaksoil sl from Oregon, differing in sexuality.

In this chapter we describe P. taxon Oaksoil from Oregon as a new sterile species

Phytophthora obrutafolium sp. nov., and characterize its physiology, and morphology. 155

We compare growth-temperature relationships between P. obrutafolium sp. nov., P. bilorbang, and another closely related species Phytophthora gonapodyides H.E.

Petersen (Buisman). Then we examine characteristics that differentiate P. obrutafolium from its closest known relatives including differences in haplotype to the closely related

P. bilorbang and P. taxon Oaksoil ss. The ecology of the new species is then examined.

The known distribution of Phytophthora obrutafolium is summarized. The apparent abundance of P. obrutafolium from streams was again examined, and is described, as well as a potential relationship with red alder stream debris, and seasonality compared to other Phytophthora species. We also examined whether zoospores could colonize alder leaves floated in stream water.

156

METHODS

Survey

As a part of a survey of alder health in Oregon eighty-eight 100 meter by 10 meter transects were laid out and sampled (Figure 1). Survey methods (from 2010) are briefly described here (Details in Chapters 2, and 3). To collect Phytophthora species two

1 L water samples, five unwashed root samples, and two soil samples were collected per transect. 157

Figure 1) The 2010 alder health survey transects (red circles) in the three designated sub-regions. The Willamette Valley (wv), southern (s) and coastal (c) (Chapter 2).

Sample processing

All water samples were filtered; root and soil samples were baited (Details

Chapter 2). Filters were inverted and placed onto VARP+ media: 15 g Bactoagar, 50 mL 158

V8 stock, 10 ppm rifamycin SV sodium salt, 20 ppm Delvocid [50% natamycin salt], 200 ppm ampicillin sodium salt, 30 ppm Benlate [benomyl 50WP] with enough sterile water to make a slurry, and 50 ppm hymexazol. Filters were incubated on the medium surface for 72 h at 20 °C. All Bait leaf pieces from baited soils were cleaned and dried then partially submerged into H/2 medium: cornmeal agar, 10 ppm rifamycin SV sodium salt,

20 ppm Delvocid [50% natamycin salt], 200 ppm ampicillin sodium salt, and 25 ppm hymexazol.

Morphological characterization to genus was done on culture plates.

Morphological characters used to distinguish Phytophthora species from other fungi growing on the plates included rate of growth, branching pattern and greater refraction of light by hyphae in media when compared to Pythium species. Occasionally isolates were ambiguous (it was uncertain if they were Phytophthora or another Oomycete such as Pythium). Ambiguous isolates were grown in water culture to evaluate zoosporogenesis. If the cytoplasm moved from the sporangium into a vesicle prior to zoospore cleavage then the isolate was classed ‘not Phytophthora’ and discarded. If cytoplasm cleaved within the sporangium, typical of Phytophthora zoosporogenesis, then it was grouped with Phytophthora. For a few isolates, zoosporogenesis did not occur and these isolates were classed as uncertain identity and set aside for DNA extraction. 159

All isolates were subcultured onto CARP media: cornmeal agar, 10 ppm rifamycin

SV sodium salt, 20 ppm Delvocid [50% natamycin salt], 200 ppm ampicillin sodium salt

(Reeser et al. 2011, Chapter 1) twice. Isolates were checked for bacterial contaminants

with a dissecting microscope at 20 – 60 times variable magnification. Isolates were subcultured on CMAβ: Corn meal agar amended with β-sitosterol, and incubated for no

more than ten days. Cork bored plugs of media containing hyphae of each isolate were

transferred into two water storage tubes (Boesewinkel 1976), and one plug of each

isolate was transferred to a sterile 50 mL tube for DNA extraction.

All isolates were DNA extracted. PCR was performed in a 50 ul reaction

containing 1 ul template DNA. PCR product was sequenced using the mitochondrial

cytochrome oxidase (Cox) spacer region flanking the Cox1 and Cox2 genes (Martin &

Tooley 2003). Sanger sequencing was done by Oregon State University Center for

Genome Research and Biocomputing sequencing center. Each isolate was sequenced in

one direction with either FMPHy-8 or FMPHy-10. Sequences were aligned in Clustal X2

(Larkin et al. 2007). Exact alignment to reference Cox spacer sequences of forest

Phytophthora species recovered from Oregon previously were used to determine species. If an isolate did not match a reference sequence exactly then it was PCR amplified in the internal transcribed spacer region with primers DC6 and ITS4 (Cooke et 160 al. 2000). Amplified products were then sequenced with DC6, ITS2, ITS3 and ITS4 as sequencing primers (White et al. 1990). Contigs were assembled and the overlapping sequences were manually edited using an editing package (Staden 1996). Edited sequences were aligned with ITS reference sequences of forest Phytophthora species recovered in Oregon. Isolate reference match, species identification, location of recovery and sample type were recorded in a database for all isolates.

Physiology

A subset of P. obrutafolium isolates was randomly selected (simple random sample) from the population of stored isolates that had been recovered from western

Oregon in 2010. These randomly selected isolates formed the collection of isolates used to examine physiology, morphology and phylogeny of P. obrutafolium (Table 1). Isolates from Jung et al. (2011), Aghighi et al. (2012) and Brasier et al. (2003) were used in the phylogenetic analysis. Isolates recovered from the 2010 survey were used to compare morphology with other species in clade 6 (Table 1). Isolates from Aghighi et al. (2012) were used for the temperature growth comparison (Table 1).

161

Sims Aghighi Aghighi Aghighi Shanahan Hansen Sims VHS Sims Sims Sims Sims Sims Sims Sims Aghighi Sims Wingfield Wingfield Collector VHS Hart

USA, OR, Oak Creek Australia, WA, Warren River River Warren WA, Australia, Australia, WA, Warren River River Warren WA, Australia, Australia, WA, Warren River River Warren WA, Australia, Australia, TAS, Pine Lake Lake Pine TAS, Australia, NE France, Amance forest USA, OR, Oak Creek Australia, Manjimup WA, USA, OR, Yachats River USA, OR, Yachats River USA, OR, Cape Creek USA, Oregon, Rock Creek Fork OR, North SiuslawUSA, River River USA, OR, East Willamette Beaver Creek Fork Coast OR, USA, River Warren WA, Australia, USA, OR, Oak Creek Chile, Auraco, plantation Location Australia, Dunninup WA, Australia, Welshpool WA, species Alnus rhombifolia R. anglocandicans anglocandicans R. R. anglocandicans anglocandicans R. R. anglocandicans anglocandicans R. native forest Quercus robur Quercus A. rhombifolia native forest Alnus rubra rubra Alnus A. rubra A. rubra A. rubra A. rubra A. rubra A. rubra anglocandicans Rubus A. rhombifolia Pinus radiata radiata Pinus Host association Banksia Banksia road drainage sump bait bait sump drainage road soil soil soil soil soil soil soil soil root water needles Substrate leaf debris stream water stream water stream water stream water stream water stream water stream water stream water

sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile Sexual behavior homothallic homothallic homothallic homothallic homothallic homothallic homothallic homothallic homothallic species considered in this study

Phytophthora

2-W-1.20 2-W-1.20 JN547645 JN547645 JN547644 JN547644 JQ256375 JQ256375 HQ012850 aecjv1 2-50-R.5 HQ012879 120-W-2.18 120-W-2.18 121-W-1.8 118-W-1.7 219-W-2.6 115-W-2.13 107-W-1.2 31-W-1.9 JN547643 JN547643 2-1-S.2 HQ012880 HQ012867 JN935961 Genbank or isolateGenbank number sp. nov sp. nov sp. nov sp. nov sp. nov sp. nov sp. nov

species taxon Oaksoil ss taxon Pgchlamydo taxon Pgchlamydo gonapodyides gonapodyides bilorbang bilorbang bilorbang bilorbang bilorbang bilorbang gonapodyides lacustris lacustris lacustris pinifolia obrutafolium obrutafolium obrutafolium obrutafolium obrutafolium obrutafolium obrutafolium obrutafolium bilorbang megasperma

P. P. P. P. P. P. P. P. P. Table 1) Isolate information for Phytophthora P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. 162

Temperature optimum - Petri plates for experiments were marked with a central

X on the back of plates. CMAβ media was poured to contain a uniform amount of media in each plate and set aside to cool. From the margin of cultures grown six days, 6 mm diameter cork borer mycelial discs were cut and transferred to the center of marked media plates. CMAβ media plates with isolate discs were grown at 20 °C for 48 h to initiate growth. Plates were then marked at the edge of the growing colony and moved to incubators. Four replicates of the ten isolates were grown at seven different incubation temperatures. Mean incubator temperatures rounded to the nearest 0.5 were: 6.0 °C, 15.0 °C, 20.0 °C, 24.5 °C, 26.5 °C, 28.0 °C, 34.0 °C. Temperatures were ℃ recorded with a data logger at fifteen minutes intervals to confirm the average incubator temperature. Following a 96 h incubation period plates were removed from incubators and the growth of the colony was measured. Radial growth was determined as the average of sixteen radii for each isolate. Each radius was determined as the distance from the marked location of growth following 20 °C incubation to the outer edge of the colony crossing the X on the back of the plate.

Temperature-growth comparison – The isolates of P. obrutafolium, Phytophthora gonapodyides (isolate 2-W-1.20, Table 1) and the isolates of P. bilorbang (Table 1, genbank numbers beginning with JN) were grown at 25 °C for 96 h to examine growth differences. Following a 96 h incubation period growth was measured. Radial growth 163 was determined as it was for the temperature optimum experiment. A linear model was constructed to examine the growth response of each species following incubation. The estimated growth values from the model were used to examine statistical evidence for differences in growth between P. bilorbang, P gonapodyides and P. obrutafolium. The model was evaluated in the R statistical environment using the [lm] command (R core team 2012).

Sexual behavior- To determine if isolates were heterothallic, isolates were paired against either a Phytophthora cinnamomi A1 or A2 isolate and observed at intervals of seven, fifteen and thirty days ± two days. For indication of a homothallic species isolates were thoroughly checked in single culture for gametangial production, observed at fifteen and sixty days of growth on three media types V8S (β-sitosterol amended V8 agar), CMAβ, and CA prepared as described in (Brasier 1969).

Morphology - Cultures of isolate 31-W-1.9 were grown on (V8S). After five to seven days of growth mycelial plugs were transferred from the margins of growing colonies into petri dishes and flooded with twice filtered stream water; water was exchanged every 24 h until several sporangia had formed. Morphological features were observed with a Zeiss standard microscope under 400-630 times magnification using brightfield illumination. Sporangial size was assessed with an eyepiece micrometer. For 164

P. obrutafolium (isolate 31-W-1.9) twenty sporangia were haphazardly selected from different areas and measured. Sporangia from five plates were counted to avoid measuring the same sporangium twice. Ten haphazardly selected exit pores were measured.

To examine differences in colony morphology cultures were grown on CA for 7 days at 20 . P. obrutafolium (isolate 31-W-1.9) and P. gonapodyides (isolate 2-W-1.20),

Phytophthora℃ lacustris (isolate 2-1-S.2), and Phytophthora taxon Pgchlamydo (isolate 2

50-R.5) were grown and compared (Table 1).

Haplotype examination - Extracted DNA from the seven isolates of P. obrutafolium and isolate aecjv1 (Table 1) were PCR amplified in the mitochondrial Cox1 gene region using Cox1 primers FM85 and FM84 (Martin and Tooley 2003). The sequencing and alignment process for Cox1 is the same as outlined above for other gene regions. The P. bilorbang fasta sequences were downloaded from GenBank (Aghighi et al 2012, Table 1) along with sequences for isolates of P. lacustris, P. gonapodyides, and

P. taxon Pgchlamydo (Jung et al. 2011, Table 1). Cox 1 single nucleuotide polymorphisms

(SNP) were examined for differences in an alignment of isolates. Isolates were grouped based on similarity. 165

Phylogeny - Alignment of consensus sequences was done with ClustalX2 (Larkin

et al. 2007). The clade 6 trees include sequences retrieved from genbank that were

described in Jung et al. (2011 and Table 1), and includes P. bilorbang isolates (Aghighi et

al. 2012 and Table 1). DNA from isolate aecjv1 (Hansen and Delatour 1999) was sequenced and used in the Cox1 phylogeny. Species from clade 6 are included. P. lacustris was assigned as the outgroup. Mr. Bayes v3.2.2 (Huelsenbeck and Ronquist

2001) was used for bayesian inference of phylogeny. A general time reversible (GTR) model with gamma distributed rate variation and a proportion of invariable sites

(invgamma) was used to analyze trees. The analysis was for two independent runs of

Metropolis Coupled Markov Chain Monte Carlo (MC)³ and was run for 1 x 10⁶ generations, with 3 heated and one cold chain. Trees were sampled every 100th

generation to gather a minimum of 10,000 sample trees. Burn in was used to discard

2500 trees, resulting in 7,501 samples from each run. [Sumt Conformat] was set to

‘Simple’. Convergence diagnostics were done in two ways: by examining the resulting

average standard deviation of split frequencies (ideal values approach zero) and by

checking the potential scale reduction factor (Gelman 1992) where values were

reasonably close to one before assuming convergence. The phylogenetic trees were

generated using TreeView (Page 1996).

166

Ecology

Geography - The geographic location of P. obrutafolium recovered in 2010 was summarized to examine the distribution of the species in western Oregon (Figure 1). The number of transects in each sub-region (Figure 1) positive or not for P. obrutafolium from baits and filtered stream water was also summarized.

Association with red alder leaf debris- During the summer, fall and winter of

2011 and 2012, five red alder leaf debris (Figure 2) samples were collected in streams beneath tree canopies from two streams (Figure 3). Transects were chosen where red alder trees were the main source of leaf debris in the streams (Figure 2-3). Two 1 L water samples were collected and paired with the first and last of five leaf debris samples (collected from each transect. One transect (121) was alongside Yachats River in Lincoln County Oregon and the other (118) was alongside Cape Creek in Lane County

(Figure 3). Alder leaves were scooped by hand from the stream bottom (Figure 2). Each leaf debris sample was placed in a separate polyethylene plastic bag and labeled with transect and sample number. 167

Figure 2) Red alder leaf debris (bottom) samples were collected in streams beneath tree canopies (top left) where red alder trees were the main source of leaf debris in streams. Leaf debris pieces were cultured on Phytophthora selective media (top right), plates were incubated and checked for colonies (arrow) growing from leaf pieces. 168

Figure 3) Two transects (red arrows) were examined year round to confirm the apparent abundance of P. obrutafolium from streams and to examine a potential relationship with red alder stream debris. Transects were chosen where red alder trees were the main source of leaf debris in streams.

169

From each leaf debris sample, hole punched leaf pieces (Figure 2, upper right) were removed from the edges of brown necrotic spots and brown areas. Each leaf piece was removed from a different leaf within the sample bag. Leaf debris was mostly green in color with brown necrotic spots and brown areas. More decayed leaf material, although in sample bags, was not processed. Unidentifiable leaves were not processed.

For each leaf debris sample leaf pieces were placed and gently submerged into a single plate of Phytophthora selective VARP+ media. Plates were incubated for 72-96 h and

checked for growing colonies (Figure 2, upper right). Isolates from all sample types were

subcultured onto CARP media.

Three sampling intervals were compared: summer, fall, and winter. Subsamples were taken once per month within these intervals. The stream water was collected one time per month per stream. Intervals were grouped: July and August (summer),

September, October and November (fall), and December, January and February

(winter). Leaf debris was collected whenever it was present in large easily recoverable quantities from streams. This occurred from July - December. When leaf debris was absent, there was an attempt to collect stream water from similar locations, but during

high water collection was from a safe location. There was no collection from one

transect in January 2012 due to unfavorable weather conditions.

170

Morphological characterization of Phytophthora to genus was done following the first subculture onto CARP media. Isolates recovered from stream debris were selected for quantification based on unique morphology (Figure 4).

Figure 4) Four isolates from a subcultured leaf debris sample. Each isolate was selected from a different red alder leaf in stream water in western Oregon. Isolates from a sample were selected for quantification based on their unique colony morphology. Each of the four colonies pictured above was considered unique.

Stream water samples were processed using the methods from 2010. Each stream water filter culture plate was examined for countable colonies. A Phytophthora isolate (or other Oomycete) was subcultured from each countable colony. A colony was considered countable if there were between 1 and 10 colonies on a plate and each colony was easily separable from a neighbor colony. Each isolate was sub-cultured onto

CARP media. Genus level identification (grouped as either Phytophthora species or not

Phytophthora species) was done from subculture colonies. The amount of filtered 171

stream water with countable colonies for each season ranged from 610 mL to 1260 mL.

The number of Phytophthora species recovered from countable colonies was

determined after identification was confirmed from sequenced DNA and a match to a

known reference (Chapter 3).

Leaf inoculation experiments- We examined whether zoospores could colonize alder leaves placed in filter sterilized stream water after a 24-72 h incubation period. P.

obrutafolium was grown on V8S media incubated for 72 - 120 h in a dark 20

incubator. From the margin of a growing colony, five colonized agar discs were℃ punched

with a 6 mm diameter cork borer and removed. Discs were transferred to petri dishes.

Dishes containing colonized agar discs were flooded with 2u-filter sterilized stream

water. Flooded plates were incubated in the dark for up to 72 h at 18 plates were

checked at 24 h intervals and stream water was replaced. Once discs contained℃; several

sporangia per disc (>10), these plates were selected. After zoosporogenesis, water containing zoospores was poured in a petri dish with clean 2u-filter sterilized stream

water. Red alder trees were grown outdoors at Oregon State University for experiments.

Alder leaves were selected with no visible damage and were carefully washed in tap

water and gently patted dry. Whole leaves were floated over plates containing

zoospores. At 24 h intervals leaves were examined for the production of hyphae and

sporangia from leaf margins and petioles. Sporangia and hyphae arising from leaves 172 were imaged with a Zeiss microscope using brightfield microscopy and fluorescence microscopy techniques. For fluorescence microscopy leaf pieces were cut and placed in concave microscope slides and stained with DAPI.

173

RESULTS

Physiology Temperature optimum - Isolates grew slowly at 6 °C and 28 °C (Figure 5). The optimal growth temperature was between 24.5 and 26.5 °C. Isolates incubated at 34 °C did not grow, nor did they resume growth in a 20 °C incubator. Isolates incubated at all other temperatures continued growth at 20 °C.

15 15 5

12.5 12.5 0

1 5 10 10

0 7.5 7.5

2 5

5 5

Average radial growth (mm) growth radial Average 3 0

2.5 2.5 3

0 0 5 10 15 20 25 30 35

Temperature( )

Figure 5) Average radial growth of P. obrutafolium after 9℃6 hours of incubation at different temperatures. Error bars represent ± 1 standard error. 174

Temperature-growth comparison – The growth response following 96 h incubation of P. obrutafolium, P. gonapodyides and P. bilorbang was compared. P. obrutafolium grew the slowest (Figure 6, mean radial growth = 12 mm), significantly slower than P. bilorbang (p-value <-0.001, mean radial growth = 14 mm). P.

gonapodyides grew the fastest (Figure 6, mean radial growth = 17 mm) faster than P.

bilorbang (p-value = 0.04) and P. obrutafolium 18 16

14 (mm) (mm)

12 aver_grwth_mm 10 Mean radial growth growth radial Mean 8 6

P. bilorbang P. gonapodyides P. obrutafolium

Figure 6) Mean radial growth of P. obrutafolium, P. gonapodyides and P. bilorbang on CA media following a 96 h incubation period.

175

Sexual behavior- Isolates of P. obrutafolium failed to produce gametangia in plated pair tests with mating type testers of the heterothallic species P. cinnamomi, or

in single culture. The failure to produce gametangia in plated pair tests and single

culture indicates P. obrutafolium is sexually sterile.

176

Taxonomy Phytophthora obrutafolium sp. nov

Holotype. ISOLATE: 31-W-1.9. OSC______, dried culture from OSU ______, recovered on September 23, 2010 from stream water in Lane County Oregon collected beneath a red alder canopy (43.59416°, - 123.03407°).

GENBANK______, MYCOBANK______. Figure 7.

Etymology -“ obrutafolium” folium for leaf and obruta for debris. Named after the close association with red alder leaf debris that accumulates in streams in riparian ecosystems in western Oregon.

DESCRIPTION

Sporangia of Phytophthora obrutafolium sp. nov. isolate 31-W-1.9 ranged in length from 41-61u with average length 53u. Observed sporangia ranged in breadth from 27-40u with the average breadth 31u. Exit pore diameter ranged from 9-13u with the average diameter being 11u. Hyphal branch points were observed with hyphal swellings (Figure 7, upper left arrow). Papillae on sporangia were inconspicuous (Figure

7, upper right arrow). Sporangia were non-caducous. Sporangial shape was generally ovoid (Figure 7, bottom center) to obpyriform (Figure 7, inset), with ovoid sporangia 177

occurring most frequently. Obpyriform sporangia had bulbous ends (Figure 7, inset).

Sporangia proliferated in various ways; internally extended sporangiophores (Figure 7

bottom left), internally nested sporangia (Figure 7, bottom right), and externally

proliferated sporangia were observed (Figure 7, upper right).

Figure 7) P. obrutafolium sp. nov. sporangia and hyphae. Swollen hyphal branch point (upper left), external proliferation of a sporangium (upper right). From left to right (bottom): internal extended proliferating growth, ovoid sporangium and, internal nested proliferation of a sporangium. Obpyriform sporangium with bulbous tip (inset). Bar = 50 u.

178

Differences in colony morphology of P. obrutafolium sp. nov. (isolate 31-W-1.9) from other closely related Phytophthora species were observed on CA after seven days

of growth incubated at 20 (Figure 8). P. obrutafolium sp. nov. grew much slower on

CA compared to P. taxon Pgchlamydo℃ (isolate 2-50-R.5), P. lacustris (isolate 2-1-S.2), and

P. gonapodyides (isolate 2-W-1.20).

Figure 8) The colony growth of P. obrutafolium sp. nov. (isolate 31-W-1.9 upper left) was much slower compared to other closely related species. P. taxon Pgchlamydo (isolate 2-50-R.5, upper right) P. lacustris (isolate 2-1-S.2, bottom left), and P. gonapodyides (isolate 2-W-1.20, bottom right).

179

Haplotype examination

Seven isolates of P. obrutafolium sp. nov., the original isolate of P. taxon Oaksoil,

aecjvi, (Brasier et al. 2003, Hansen and Delatour 1999), and four isolates of P. bilorbang

(Aghighi et al. 2012) were compared for haplotype single nucleuotide polymorphism

(SNP) differences using aligned sequences of the mitochondrial Cox1 gene region (Table

2). Isolates were grouped based on similarity and species name and genbank/isolate

number was colored to identify matching groups. SNP differences unique to a group

were colored. P. obrutafolium sp. nov. comprised three haplotypes, the greatest

difference within the species was two base pairs (compare 121-W-1.8 to 115-W-2.13, at position 114 and 225).There were no differences between any of the P. bilorbang isolates. P. bilorbang differed by at least four base pairs from any other species. There were seven base pair differences between P. bilorbang and aecjv1. This isolate was a unique haplotype with at least three base pair differences from any other species isolate. The greatest similarity between isolates of two different species was between aecjv1 and 120-W-2.18 which differed by three base pairs.

180

Table 2) Cox 1 SNP chart comparing haplotype differences for three similar species.

cytochrome oxidase subunit 1 SNP chart Genbank or isolate Species number 57 72 93 114 135 177 225 672 684 P. bilorbang JN547643 C T A A A C T G T P. bilorbang JQ256375 C T A A A C T G T P. bilorbang JN547644 C T A A A C T G T P. bilorbang JN547645 C T A A A C T G T P. taxon Oaksoil ss aecjv1 T C T A C T T T C P. obrutafolium 121-W-1.8 C T T A C C C T C P. obrutafolium 118-W-1.7 C T T A C C C T C P. obrutafolium 120-W-2.18 C T T A C C T T C P. obrutafolium 219-W-2.6 C T T A C C T T C P. obrutafolium 115-W-2.13 C T T G C C T T C P. obrutafolium 107-W-1.2 C T T G C C T T C P. obrutafolium 31-W-1.9 C T T G C C T T C

181

Phylogeny

In the Cox1 phylogeny P. obrutafolium sp. nov. isolates from western Oregon streams were separated from the blackberry pathogen P. bilorbang isolates from

Western Australia, and the isolate from French oak forests (Hansen and Delatour 1999) first identified as P. taxon Oaksoil (Brasier et al. 2003) (Figure 9). Seven other isolates comprising five other species (Jung et al. 2011, Aghighi et al. 2012) were included in the analysis but are not pictured (Figure 9) because they separated on distant branches.

Cox1 Genbank numbers for these isolates were: HQ012850, AY129175, HQ012867,

HQ012879, HQ012878, JN935961, HQ12880 (Table1).

Figure 9) Cox1 phylogeny showing the three closely related species.

182

Ecology

Geography - In the 2010 survey P. obrutafolium was recovered from 58 of 88 transects (Figure 10). More transects had positive isolation from stream water than from the rhizosphere. (Table 3). P. obrutafolium was identified across all of western

Oregon (Figure 10).

Figure 10) Map showing the locations of transects in western Oregon that were positive for P. obrutafolium. 183

Table 3) In western Oregon riparian ecosystems the number of transects in each sub-region positive or not for P. obrutafolium from baits and filtered stream water.

Presence/Absence sub-region P. obrutafolium bait water Willamette Valley + 5 16 Willamette Valley - 28 17 Coastal_ + 5 27 Coastal_ - 25 3 Southern Oregon + 5 9 Southern Oregon - 20 16 184

More isolates per liter of stream water were recovered for P. obrutafolium in the summer and fall, coinciding with leaf fall and accumulation in streams (Figure 11) than in the winter. Few propagules were collected in the streams during the winter after leaf debris had washed away. Phytophthora species were easily and consistently isolated from leaf debris (Figure 2 upper right).

Other Phytophthora species P. obrutafolium

Figure 11) Isolates per liter of P. obrutafolium and other Phytophthora species from stream water for three season. Data is from two streams in western Oregon riparian ecosystems. 185

More than half (84/160) of the Phytophthora isolates from leaf debris were P. obrutafolium. The cumulative percentage of P. obrutafolium recovered over the same period of time (summer, fall, and winter) and from the same locations (Cape Creek and

Yachats River) from stream water and leaf debris was comparable (57% and 53% respectively, Figure 12 a-b).

(a)

7% 5% 10% P. obrutafolium P. gonapodyides P. taxon Pgchlamydo P. siskiyouensis P. pseudosyringae 21% 57%

(b) 2% 1% 4% 2% 19% P. obrutafolium P. gonapodyides P. taxon Pgchlamydo P. lacustris P. siskiyouensis P. pseudosyringae 19% unknown 53%

Figure 12) Western Oregon recovery of Phytophthora. (a) - Phytophthora species recovered from streams associated with red alder. (b) - Phytophthora species recovered from red alder leaf debris. 186

Leaf Inoculations - Sporangia formed on detached alder leaves floated on sterilized creek water infested with P. obrutafolium zoospores. Sporangia and hyphae were observed using standard microscopy (Figure 13, left) and florescence microscopy (Figure 13, right).

Zoosporogenesis was observed from artificially inoculated leaves and can be inferred from the empty sporangium in Figure 13 (left).

Figure 13) Red alder leaves artificially inoculated with P. obrutafolium zoospores. (Left) An empty sporangium indicating zoosporegenesis has occurred; sporangiophore and hyphae extend from the leaf surface. (Top right) Above the leaf surface several sporangia were observed. (Bottom right) A sporangiophore with sporangium was observed attached to the leaf surface.

187

DISCUSSION

The optimal growth temperature for P. obrutafolium sp. nov., averaging 25 °C, was similar to many Phytophthora species in clade 6 (Brasier et al .2003, Jung et al

2011). However, at 25 °C P. obrutafolium grew more slowly on CA media than the two closely related species P. bilorbang and P. gonapodyides. It also grew much slower on

CA when compared to other closely related clade 6 species that are more genetically distant than P. bilorbang. P. obrutafolium also had a unique haplotype when compared to its closest known relatives: the blackberry pathogen P. bilorbang from Western

Australia and the original P. taxon Oaksoil isolate aecjv1 from an oak forest in France.

P. obrutafolium was widespread and seasonally abundant in western Oregon streams coincident with alder leaf fall and accumulation. P. obrutafolium appears to have an ecological niche in western Oregon as an aquatic saprophyte of alder leaves and possibly a minor pathogen of streamside red alder. If it associates with alder away from streams this is not yet known. P. obrutafolium appears to reproduce abundantly by asexual means in streams where and when red alder leaf debris is present.

DNA sequence data from the mitochondrial encoded cytochrome oxidase subunit (Cox)1 can be used to help characterize Phytophthora species (Martin and 188

Tooley 2003, Gómez-Alpizar et al. 2008, Blair et al. 2008, Jung et al. 2011, Kroon et. al

2012). Usually ITS is used to distinguish Phytophthora species but sometimes species are identical in the ITS region (Gómez-Alpizar et al. 2008). This can be a difficulty that has been recognized by others for the ITS region (Álvarez and Wendel 2003, Bailey et al.

2003, Kroon et al. 2012, Gómez-Alpizar et al. 2008). ITS does not distinguish between P. obrutafolium and its closest relatives (data not shown). However, examination of the haplotype was a successful way to separate species that not only differ in geographic distribution, but also in host association, physiology and sexual behavior.

In the 2011- 2012 survey of stream water and alder leaf debris it was found that

P. obrutafolium was as common in stream water associated with red alder as it appeared to be in the 2010 survey. Survey efforts strongly suggest that P. obrutafolium is common in streams in association with red alder. From the 2011- 2012 survey of stream water P. obrutafolium composed a similar but slightly greater proportion of recovered Phytophthora isolates when compared to stream water recovery in 2010

(Chapter 3, 57% and 52% respectively).

Sporulation from debris was the factor targeted as driving the P. obrutafolium abundance in stream water in association with red alder. Evidence from field recovery supports the association with red alder leaf debris and laboratory experiments support 189 that sporulation from red alder leaf debris is possible. P. obrutafolium was most commonly recovered from stream water during the fall when leaves were accumulating in the stream (Figure 2).

190

CONCLUSIONS

• P. obrutafolium sp. nov. optimal temperature for growth was between 24.5 and

26.5 °C.

• P. obrutafolium sp. nov. grows slower on CA than P. bilorbang or P.

gonapodyides.

• P. obrutafolium sp. nov. from western Oregon has a unique haplotype compared

to its closet known relatives: the blackberry pathogen P. bilorbang from Western

Australia, and P. taxon Oaksoil ss from an oak forest in France.

• P. obrutafolium was widespread and seasonally abundant in western Oregon

streams associated with red alder coincident with alder leaf fall and

accumulation.

• P. obrutafolium is seasonally abundant in western Oregon

• Detached leaf colonization and sporulation experiments with P. obrutafolium

were successful.

• Data from western Oregon support an ecological role for P. obrutafolium as an

aquatic alder leaf saprophyte.

191

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Chapter 6: Dissertation Conclusions

MOTIVATION In 2009, an unusual amount of alder (Alnus Miller (Betulaceae)) tree mortality was observed along the Smith River in Douglas County, Oregon. In the same area living alder trees with bleeding cankers and canopy dieback were reported. Further observations in western Oregon suggested that similar mortality and canopy dieback might not be confined to one area. A systematic examination of alder trees in western

Oregon had not been done at the time. Concerns about the health of alder trees were amplified in Oregon because of recent disease problems of alder in other places. Around the world, outside of western Oregon, pathologists were contending with diseases of alder trees caused by various organisms, including species of Phytophthora de Bary,

Cytospora Ehrenb., and Melampsoridium Kleb. In the early 1990’s, an invasive

pathogenic Phytophthora species was found killing alder trees in Southern Britain (Gibbs

1995). The most virulent subspecies, P. alni subsp. alni Brasier & S. A. Kirk (Brasier et al.

2003), had affected or killed an estimated 15% of alder across southern Britain (Webber

et al. 2004). There were no known Phytophthora disease problems of alder in natural

ecosystems in the United States at the time. P. alni subsp. uniformis Brasier & S. A. Kirk

was later found in riparian soils in Alaska in association with Alnus incana (L.) Moench

(Adams et al. 2008) but was not associated with any dramatic disease there. Only one

Phytophthora disease of alder had been described in the United State and this was on 195

planted Alnus cordata (Lois.) Duby Italian alder trees California. It was a concern that

Phytophthora alni or another Phytophthora species could be causing disease in western

Oregon and that disease would go undetected because riparian areas where most alder

occur are not normally monitored for disease problems. This was especially

disconcerting because red alder (Alnus rubra Bongard), the most common alder species in Oregon, is an economically viable hardwood timber species, and provides irreplaceable ecological services in riparian ecosystems.

DISSERTATION GOAL AND OBJECTIVES

Damage surveys began in 2010 on alder trees in western Oregon riparian ecosystems and continued through 2012. The goal was to gather baseline data on damage associated with alder trees in western Oregon riparian ecosystems. The first objective was to record and assess damage to alder trees. The second objective was to determine and evaluate Phytophthora species in western Oregon riparian alder ecosystems, including streams, the rhizosphere (soil and unwashed roots), and tree roots (washed and surface sterilized), while monitoring for Phytophthora alni. The third objective was to evaluate damage caused by Phytophthora infection of alder in western

Oregon. The fourth objective was to examine ecological associations of Phytophthora to alder trees in western Oregon riparian ecosystems. The fist objective was addressed broadly in Chapter two, in which canopy dieback symptoms were grouped and analyzed according to damage type: (1) pathogen, (2) insect, or (3) wound. The second objective 196 was addressed in chapter three in which the assemblage of Phytophthora species from western Oregon riparian ecosystems was examined and compared. The third objective was addressed in Chapter 4, in which Koch’s Postulates were evaluated for three key

Phytophthora species, and alder canker disease in the United States is described. In chapter 5 the fourth objective is addressed in the examination of the ecological role of the most abundant Phytophthora species from streams and a new species is described

P. obrutafolium sp. nov.

CHAPTER 2 In chapter two, the damage survey of alder trees in western Oregon riparian ecosystems is described. Damage from pathogens, insects and wounds was examined in relation to alder tree canopy dieback. Many of the same damaging agents that have been recognized in the past on alder trees in the western United States were also found damaging alder trees in western Oregon riparian ecosystems. The data indicated that some of the damage was from the alder flea beetle, sawflies, flood debris, and foliar pathogens. Foliage pathogens, only previously recognized as being important in a nursery setting or in the canopy understory, may be important at least in coastal areas to red alder in particular. Foliage insects, including many introduced species were causing damage to alder trees, but this is not new and for some species, it has been suggested damage is episodic. Wounding to alder trees in western Oregon riparian ecosystems was not an important damaging factor in relation to canopy dieback but was 197

important in relation to stem decay and many trees had wounds. It was found that

Phytophthora cankers were associated more than other damaging agents with canopy

dieback, and were tallied on 2.5% of alder trees. Phytophthora siskiyouensis was

isolated from bole cankers of red alder and white alder trees. The most virulent subspecies of the alder Phytophthora damaging alders in Europe, Phytophthora alni subsp. alni, was not identified in western Oregon. It was concluded that Phytophthora canker symptoms do occur on alder in natural stands in the Americas; cankers were widespread in western Oregon riparian ecosystems in stands containing trees with canopy dieback.

CHAPTER 3 The goal in this chapter was to describe the assemblage of Phytophthora species isolated and identified from riparian alder ecosystems in western Oregon. Phytophthora species and clade composition were compared from the rhizosphere and from the stream water. Phytophthora species and clade composition were also compared from direct isolation of diseased woody and fine alder roots. Western Oregon riparian ecosystems were evaluated for the presence of Phytophthora alni from all substrates. It was determined whether Phytophthora siskiyouensis or any other Phytophthora species was associated with alder from all the examined substrates or recovered from diseased alder roots. It was concluded that although Phytophthora species are widespread and diverse in association with alder in western Oregon only three species were associated 198 with all substrates: P. siskiyouensis, P. gonapodyides and P. taxon Pgchlamydo.

Phytophthora alni spp. uniformis was isolated from alder roots at two sites. P. taxon

Oaksoil was the most common species from stream water and probably was not an important pathogen of alder because it was rarely isolated from the rhizosphere, and was not isolated from alder roots.

CHAPTER 4 In this chapter, damage caused by Phytophthora infection of alder was evaluated. The pathogenicity of P. siskiyouensis, P. alni subspecies uniformis and P. taxon Oaksoil to alder species from western Oregon was tested. Koch’s Postulates were evaluated for these three Phytophthora species. A bole canker caused by Phytophthora,

Phytophthora canker of alder, was described. It was found that both P. siskiyouensis and

P. alni caused large cankers on inoculated alder seedlings, but that P. taxon Oaksoil caused cankers not significantly larger than negative controls. It was concluded that although both P. siskiyouensis and P. alni were able to cause large cankers in pathogenicity tests P. siskiyouensis is the likely causal agent of Phytophthora canker in western Oregon because incidence of alder disease tied to P. alni was low. In Chapter three it was found that the most common Phytophthora species in western Oregon in association with alder was P. taxon Oaksoil. The relationship was reevaluated in this chapter in terms of the prevalence of canopy dieback in alder stands in terms of the two

Phytophthora species that were recovered on a regular basis from the alder stands, P. 199

taxon Oaksoil and P. siskiyouensis. It was found that although P. taxon Oaksoil was the

most common species the prevalence of canopy dieback was greater in stands with P.

siskiyouensis than in stands with P. taxon Oaksoil alone. In summary, canopy dieback

was more prevalent in riparian alder trees from transects with P. siskiyouensis (70%)

than from transects with P. taxon Oaksoil but without P. siskiyouensis (35%).

Phytophthora canker of alder found in western Oregon riparian ecosystems in the

United States was attributed to P. siskiyouensis.

CHAPTER 5

In this chapter, a new species, Phytophthora obrutafolium sp. nov., is described.

Formerly referred to in this dissertation as Phytophthora taxon Oaksoil, it was found to

differ from P. taxon Oaksoil sensu stricto (also referred to as isolate aecjv1). P.

obrutafolium also differed from another closely related species P. bilorbang. P.

obrutafolium sp. nov. Optimal temperature for growth of P. obrutafolium was between

24.5 and 26.5°C. P. obrutafolium grew slower on CA than both P. bilorbang and P.

gonapodyides. P. obrutafolium sp. nov. from western Oregon had a unique haplotype in

the Cox 1 gene region compared to its closest known relatives, the blackberry pathogen

P. bilorbang from Western Australia, and P. taxon Oaksoil ss from French oak forests. P.

obrutafolium was widespread and seasonally abundant in western Oregon streams

associated with red alder. Detached leaf colonization and sporulation experiments with 200

P. obrutafolium were successful. This species was seasonally abundant in western

Oregon coincident with alder leaf fall and accumulation. Data from western Oregon support an ecological role for P. obrutafolium as an aquatic leaf saprotroph of alder.

SUMMARY

In summary, other agents besides Phytophthora can damage alder trees in western Oregon. Many Phytophthora species associate with alder in western Oregon, but not all of them are important damaging agents. In fact, the main role of P. obrutafolium in relation to red alder was as an aquatic alder leaf saprotroph. However,

Phytophthora canker of alder is a serious disease of alder trees, highly correlated to canopy dieback, and it was widespread in western Oregon. In the United States, above ground cankers have only been found to be caused by P. siskiyouensis. Alder trees with

Phytophthora canker of alder are likely to have canopy dieback and Phytophthora infected roots.

201

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