Phytophthora Community Survey at the Tualatin River Farm

The Pacific University 2024 Plant Pathology class extensively studied Phytophthora species as a Course-Based Research Experience (CURE). In particular, we were interested in investigating the Phytophthora community structure at the Tualatin River Farm in Hillsboro, Oregon given the land-use history of the site. 

The Tualatin River Farm is owned by Clean Water Services, the company that manages and treats water resources in the Tualatin River Watershed in Washington County, Oregon. Because the site has an agricultural history of tree and rhododendron farming, there is a potential that Phytophthora species may have already been introduced through host species. Currently, the Tualatin River Farm grows plants for restoration efforts, so there is an additional possibility of accidental introductions of Phytophthora and other oomycete species that are coming and going through the site. 

Twelve students were divided into groups to investigate 2 primary sites: 

1. The upper region where the noble fir monoculture once was (which includes wetbeds and the common garden)

2. The lower acreage (which includes the Tualatin River, a wetland region, and an outflow region through which water flows from the upland to the lowland area)

We had several specific goals:

1. Determining whether Phytophthora and other oomycete species were present at Tualatin River Farm
2. Identifying the specific species present and mapping where they are located
3. Determining whether oomycete species were concentrated on trails and/or unintentionally transported by human activity on trails (the movement of soil on shoes or equipment)
4. Determining the implications of the species types and locations for Clean Water Services

The Tualatin River watershed drains 712 square miles and flows through a large amount of agricultural, residential, and forested areas. From its source in the Northern Oregon Coastal Range to the Willamette River the Tualatin River goes on for 78 miles, promoting both agricultural growth and wildlife habitats (Ame, 2007).

Clean Water Services focuses on promoting the growth of the Tualatin River Watershed while also safeguarding public health by cleaning and returning water to the river free of pollutants (CWS). Providing over 600,000 people with sewer and surface water management. Clean Water Services uses the Tualatin River Farm as a nursery for native plants to use for restoration projects across the region. Prior to being used by Clean Water Services, this acreage was used as a residential space, agricultural fields, and a noble fir tree monoculture. The upper fields of this land were for agricultural use and the lower acreage of this space, also known as the lower floodplain, were agriculturally barren. 

Non-native phytopathogens, or plant pathogens, pose an extreme threat to the biodiversity and functioning of natural ecosystems when introduced. Several different pathways may cause accidental introductions, but one pathway that has been shown to introduce invasive phytopathogens is restoration plantings, specifically using infected plant material for restoration projects (Donald et al., 2021; Sims and Garbelotto, 2021). 

One genus of plant pathogens that has support for introduction via restoration projects is Phytophthora (Donald et al., 2021; Sims and Garbelotto, 2021). This pathogen genus belongs to a phylogenetic lineage of fungus-like organisms, called oomycetes (Hansen, 2007). Species within Phytophthora are characterized by their filamentous hyphae and multiple spore stages, including survival and swimming spore stages, contributing to their ease of dispersal and persistence in the environment. There are currently over 325 described species of Phytophthora classified within 12 phylogenetic clades (Matsiakh and Menkis, 2023). 

Phytophthora ramorum and Phytophthora lateralis are two locally-relevant species of concern within the genus. P. ramorum has a broad host range, but is most known for and primarily affects oak trees; it causes the diseases ‘Sudden Oak Death’ (SOD) and ‘Ramorum Blight’ (Rizzo et al., 2002). P. ramorum is currently classified by the USDA, in the Pacific Northwest, as a quarantine pathogen, meaning that plant material transport to and from quarantined areas is monitored under regulatory strategies. P. lateralis despite a much narrower host range, has largely affected Port-Orford-Cedars (POC) in the Pacific Northwest, as the pathogen is the causal agent of ‘POC Root Disease’ (USDA). Both Phytophthora species are non-native and responsible for detrimental ecological impacts in the Pacific Northwest and California. 

Methods to survey for oomycetes, particularly Phytophthora, are quite simple. Soil baiting, bucket baiting, and stream baiting are all techniques that can be used to capture species of Phytophthora. Each technique employs a combination of known host tissue and water, which baits the swimming zoospore stage.

Phytophthora sampling locations at Tualatin River Farms were chosen based on proximity to outside water sources (river, wetland, or wetbeds), restoration sites, or areas with symptomatic foliar hosts. For each location, the soil moisture and temperature were recorded. 

To collect soil, surface debris was removed before digging with sanitized trowels. Gallon-sized zip-top bags were filled up ¼-⅓ with soil containing plant roots. Each bag was labeled with the location, date, and lab group to distinguish individual samples. Multiple samples were taken from each location, so trowels were sanitized with bleach towels before sampling the next location.

At the lab, soil sample bags were left open to allow gas exchange. One week later, 100 grams of soil was taken from each sample, placed in plastic containers, and filled with 200 ml of deionized water. Floating debris was skimmed from the surface, and susceptible host tissue, rhododendron leaves or rose petals, were cultured to confirm oomycete absence before floating on top of water as bait (Shew and Benson, 1982). After one week, floating tissue baits were observed for any necrotic lesions or symptoms that could be used to isolate cultures. 

For bucket bait sampling, one bucket was deployed per location. Standard five-gallon plastic buckets were filled with well water and rhododendron leaves floated on the surface. Mesh lining and bungee cords covered the top to prevent foreign material contamination (Hansen et al., 2008). Buckets were placed under symptomatic hosts, primarily Oregon grape and rhododendrons, and collected two weeks later.

To collect the sample, the mesh lining and bungee cords were taken off, leaves were removed and placed into zip-top bags, and well water was dumped on site. Leaves from bucket baits were brought to the lab and observed for any necrotic lesions or symptoms that could be used to isolate cultures.

Bait bags were crafted using plastic mesh fabric to facilitate water flow, incorporating a grommet for attachment. Rhododendron leaves were washed with detergent and rinsed in 70% ethanol. Four leaves were placed within each bait bag. In the field, anchors were positioned near targeted areas of interest and affixed to the bait bags using rope. Water baits were distributed across the nursery's wetland, river, wet bed, and pond. Bait bags floated for 7 days before retrieval, followed by subsequent laboratory analysis. To monitor temperature variations, sinking thermometers were affixed to one water bait per site to record initial and final temperatures. Bait bags were stored at the lab for 1 day at 4°C.

Bosc pears, free of wounds, were selected and sterilized with ethanol. Pears were placed in wildlife proof fishing bait cages were modified to be used to deploy pear baits for this study. These pear baits were anchored to some of the same stakes utilized for our water baits. The pear setups were left to float undisturbed for 6 days. Pear bait setups were retrieved and stored at the lab for 1 day at 4°C.

To test our leaf and pear baits for Phytophthora we conducted a Lateral Flow test using ImmunoStrip® for Phytophthora species (Agdia INC., Elkhart, IN). We modified the protocol by using a mortar and pestle to grind up a piece of leaf tissue with the Extraction Buffer to ensure sufficient tissue disruption. For pear tissues, a piece of infected tissue can be placed directly into the Extraction Buffer vial and shaken vigorously to break up the tissue. Once we have a liquid mixture, we use a sterile pipette and place 3 drops of the mixture into the test strip. The test was read after 10 minutes. A positive test had two lines, whereas a negative had 1 line and a failed test would have 0 lines. 

To further confirm that our specimens are infected with Phytophthora, we used a DAS-ELISA PathoScreen® test for Phytophthora species (Agdia INC., Elkhart, IN). We cut a small piece of infected leaf tissue and weighed its mass. Then we added a 1:10 ratio of GEB2 buffer with the plant tissue. For example, 0.15 g of sample will use 1.5 mL of GEB2. We grinded the sample using a mortar and pestle to create a green juice mixture. From this mixture, we extracted 300 μL into a microcentrifuge tube for each sample. Each well was filled with 100 μL of 1X PBST to hydrate the wells, soaking for 4 minutes and tapping it upside down to empty the wells. Next, add 100 μL of each sample and repeat 2 more times for a total of 3 wells for each sample. Positive and negative controls were also tripled, filling negative wells with water. The plate is incubated in a humid box overnight at 2 - 8 °C. Next, we will prepare the enzyme conjugate and dilute it using 1X RUB6 buffer to make 100 μL of dilute for each well. Using the 1X PBST buffer, we washed the plate 8 times, then added 100 μL of diluted enzyme conjugate into each well. The plate was incubated at room temperature for 2 hours. We washed the plate again 8 times using 1X PBST buffer to wash out the enzyme conjugate. After the wash, we pipette 100 μL of diluted PNP into each well and incubate without light for 1 hour at room temperature. We observed the colors of the wells and used a spectrophotometer to measure the O.D. values. Positive O.D. values (yellow) indicate the presence of a Phytophthora species.

Baits were cultured in petri dishes. Preventing cross contamination during the culturing process was prioritized. Lab bench was split into designated “clean” and “dirty” zones. All tools including but not limited to forceps, scissors, scalpels etc were sanitized between contact with samples using an incinerator. Each culture following the following procedure: 

1. Sanitation: Baits (rhododendron leaves, rose petals, pear, etc) were first sanitized using deionized water and bleach.
2. Sample Collection: Tissue collected for plating was taken from the margins between symptomatic and healthy tissues. If no symptoms were clearly visible, tissue was collected from leaf tips and petioles (when applicable). 
3. Plating Samples: four 3x3mm were collected for each culture. Tissue samples were evenly distributed around the edges of the petri dish. In the case of pear baits, an additional 5th tissue sample was placed in the center. 

A number of cultures required re-plating. Re-plating method again followed the hyphal-tip protocol, using populated agar from initial culture to re-plate. 

DNA was extracted via a silica-based DNA isolation technique, using the QIAGEN DNeasy Plant Mini Kit. Plant tissue came from pear, water, soil, and bucket baits, and initially lysed using a mortar and pestle. Our extraction method used the enzymes AP1, and RNase A, followed by incubation at 65°C for 10 minutes. Finally, Buffer AW1, AW2, and AE were used to wash and isolate infected plant tissue, followed by incubation at room temperature for 5 minutes.

To analyze our samples for the presence of Phytophthora species, Polymerase chain reaction (PCR) tests and agarose gel electrophoresis were performed. The primers used were ITS4 and ITS6. MyTaq, from Meridian, was the DNA polymerase employed in the PCR master mixes. ITS6 and ITS4 were used to distinguish Phytophthora species DNA, which is represented by a band between 500-900 base pairs. Samples with bands matching the expected values for ITS6 or ITS4 were scored as positive for the presence of Phytophthora, the absence of this band was scored as negative. For this experiment, 1.5% agarose gels were made using 1xTAE buffer, agarose, and gel red. The ladder used was Biolabs 100bp DNA Ladder. PCR product was purified and sequenced (Eurofins Genomics, Louisville, KY). Sequences were analyzed using the NCBI BLAST tool in the Phytophthora database.

• Both ELISAs and Lateral Flow tests returned positive results for Phytophthora
• P. ramorum was not detected
• The most common Phytophthora species isolated were Phytophthora citricola and Phytophthora chlamydospora; Elongisporangium anandrum and Globisporangium intermedium were the most common species isolate within Pythiaceae family.

Our analyses and data returned many species of Phytophthora, other Oomycetes, and True Fungi, but to confirm these species, we would like to purify the PCR product again using the ITS6 primers to build higher fidelity complementary sequences. 

The majority of our Phytophthora species isolates are classified within Clade 2. Clade 2, divided into subclades a-e, is the largest of the 12 documented clades of Phytophthora; most of its species are described as virulent phytopathogens, and all species occur in terrestrial habitats (Abad et al., 2023; Yang et al., 2017). In humid environments, however, some species within the clade have adapted to aerial survival capabilities, contributing to further infection of aboveground plant parts, such as leaves, stems, and fruits (Abad et al., 2023).  

Other Phytophthora species isolated are classified within Clade 6. Clade 6 is divided into subclades a-d (Yang et al., 2017). A large number of the species within Clade 6b are aquatic specialists and are described as weak pathogens Abad et al., 2023). However, there are a few exceptions within the subclade, one of which is one species isolated from TRF, Phytophthora megasperma. P. megasperma was first identified in 1931 and parasitizes many hosts within the Fabaceae family, typically able to be found on all parts of the plant (Abad et al., 2023; Identity Technology Program). Only one isolate returned a hit for this species. The other species isolated within this clade is described later.

The most common Phytophthora species isolated according to the Krona plot produced from our sequencing data were Phytophthora citricola and Phytophthora chlamydospora. P. citricola (Clade 2c) has a wide host range, some of which include rhododendrons, pine, and true firs; it is commonly associated with container and field nurseries using overhead systems for irrigation (OSU Dept. of Horticulture). P. chlamydospora (Clade 6) also has a wide host range, capable of infecting many species with both hard and soft wood. This particular species is found globally in wet soil and waterways, and it has been described as a pathogen of riparian species and nursery stock (Hansen et al., 2018). 

One important species of True Fungi that was isolated from the forest area was Neonectria punicea. N. punicea has a wide host range, most commonly associated with ash and beech trees, and parasitizes endophytically. This fungal pathogen has not shown direct penetration capabilities, but upon entry through natural openings or prior wounds into a host, it can cause cankers and necrosis in juvenile ash trees (Karadžić et al., 2020).

Phytophthora ramorum, classified within Clade 8, was not detected or isolated, which was the species of utmost importance and concern for this project. This suggests that the USDA quarantine classification and combined regulations have been successful in limiting the spread to novel counties. However, P. ramorum still poses a great risk to native plants and broader ecosystems in Oregon, including Washington County; continued surveillance is needed.

This project was the first plant pathogen and Phytophthora diversity index conducted at the Tualatin River Farm. More studies like this are necessary to inform key stakeholders about limiting invasive species' accidental introduction with restoration efforts.

1. Re-Run PCR product with ITS6 primers to yield complementary sequences for phylogenetic analysis. 
2. Determine if further site inquiry is desired, and develop a sampling schedule that would help Clean Water Services understand the distribution and diversity of existing Phytophthora species on site. 
3. Develop site-specific phytosanitation measures that might limit the movement of these pathogens off-site.
4. If desired, conduct pathogenicity testing with specific isolates with plant species that are grown at the Tualatin River Farm. This would help us understand the potential risk to plant health related to the Phytophthora species on site. 
5. Analysis of other sites is needed to determine if these pathogens are limited to the Tualatin River Farm, or if they are more widely distributed in the Tualatin Watershed. 

As a class we would like to give our thanks to all the following parties who allowed this project to be possible: Clean Water Services; specifically Vance Kimball, and Randy Lawrence for your hospitality and enthusiasm for our experiment. We would also like to thank Dr. Joey Hulbert and Dr. Marianne Elliot, with Washington State University Puyallup Research and Extension Center, for their mentorship on our experimental design. Lastly, we’d also like to thank the Departments of Environmental Science and Biology at Pacific University. Special shout out to Juniper Grimes and Miranda Karson for supporting our lab experience.

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