Dr. Marianne Elliott returned to Seward Park on Tuesday April 4th to collect a few dozen soil samples for subsequent phytophthora testing at her lab at WSU Puyallup. We collected from healthy, dead and intermediate locations. She also collected leachate samples prepared by David George and me, twelve bags for six species of potted plants, to further explore the possibility that Phytophora infection is being introduced from native plant nurseries.
Marianne last visited the forest in November 2015. She remarked on the extent and severity of the sword fern die-off.
Project Title: Examination of the role of soilborne plant pathogens in restored, undisturbed, and dieoff sites in the decline of western sword fern, Polystichum munitum, in Seward Park
Investigators: Marianne Elliott1, Katie Coats, Lucy Rollins, and Jenny Glass, Puyallup Research and Extension Center, Washington State University, 2606 West Pioneer, Puyallup, WA 98371; Paul Shannon, Seattle, WA; 1melliott2@wsu.edu
Project Summary: The decline of the western sword fern (Polystichum munitum) in Seward Park has been observed over several years, and in some areas of the Park, western sword ferns have completely disappeared. Moreover, the spatial extent of the decline of the western sword fern appears to be expanding, suggesting a possible biotic cause of the decline. The pattern of the dieoff resembles that of a soilborne pathogen such as Phytophthora.
The genus Phytophthora contains some of the most destructive plant pathogens that are known. These include diseases of food crops, such as P. infestans, the cause of potato late blight, and forest declines, such as that caused by P. cinnamomi. The only Phytophthora species reported on sword fern is P. cinnamomi, which was found in California (Tidwell et al. 1984). Phytophthora and other plant pathogens can become introduced to a site via contaminated soil on vehicle tires, shoes, and equipment. Another means of introduction is via contaminated potting media and plant roots of nursery stock used in outplanting at restoration sites. We are presently examining this pathway of introduction in another project and are including the Seward Park site in this study.
In an earlier study in November 2015, we sampled soil, roots, and tissue from root crowns of sword ferns in an unaffected site and in "ground zero", a severely affected dieoff site. No Phytophthora, Pythium, or fungal pathogens were found on any of the samples. We re-sampled in November 2016 and had the same results by culturing. At that point quantitative PCR, a highly sensitive molecular test for the Phytophthora genus, was run and low levels of Phytophthora were detected in all samples from the soil and roots from affected and unaffected plants. This may indicate a ubiquitous non-causal Phytophthora is present. Another consideration is that the number of Phytophthora species present is not be decipherable by this assay. It is possible that the cold temperatures were not conducive to Phytophthora sporulation activity, so the next sampling took place in April 2017. Results from this sampling are given below.
In this project, we sampled for Phytophthora in soil under sword ferns that are healthy and in areas where the dieoff is occurring. We also collected samples from the root zone of plants in restored areas. These plants were planted from potted nursery stock in the past 2 years. We will conduct a comparative analysis to determine if there are differences in fungal and oomycete pathogens present among the healthy and dieoff sites. The results of this research could shed new light as to the potential cause(s) of the western sword fern decline, and potentially provide managers with new information to manage the ongoing decline.
Sampling Methods: Using the protocol developed by USDA-APHIS for P. ramorum (USDA-APHIS 2010), we collected approximately 500 g of soil beneath five plants at each of six sites in Seward Park (Figure 1). The sites included four recently planted areas ("healthy"), the "ground zero" site, and an older dieoff site. At each of the four healthy sites, soil was sampled beneath four recently planted plants and one healthy sword fern (Table 1). At the two dieoff sites, soil beneath five declining or dead sword ferns was sampled. The four sites with healthy sword ferns are located along the Lost Lake trail and the two dieoff sites are near the Sqebeqsed Trail of the Park, and are within a relatively short walking distance from each other. Soil samples were taken to WSU Puyallup and refrigerated for 3 weeks.
Analysis: All soils were baited according to the APHIS protocol (USDA-APHIS 2010). Briefly, the soil in each bag was thoroughly mixed and made to an approximate depth of about 1" in the gallon bag. About 200 ml water was added to the soil and thoroughly wetted. A Rhododendron 'Nova Zembla' leaf bait was added to each bag. Bags containing soil, water, and leaf baits were incubated at room temperature for 72 hours, after which time the leaves were removed from the bags, rinsed, and incubated on damp paper towels in sandwich-sized ziplock bags. After 4 -7 days, symptomatic areas of leaves were removed and cultured in Phytophthora-selective media. Colonies with morphological features resembling Phytophthora spp. were isolated into pure culture, where they were examined in the microscope.
Representative isolates were sequenced using the ITS region of the rDNA, covering ITS1, 5.8s, and ITS2, was amplified with primers ITS6 and ITS4 (White et al. 1990) using direct PCR (Calmin et al. 2007). The mitochondrial COX region was sequenced for further examination to distinguish Phytophthora species with similar or identical ITS sequences. Preliminary identification of species was done by querying the sequences against the GenBank database using BLAST. Identification of isolates by sequence was determined using the GenBank database as well as the Phytophthora databases Phytophthora ID (http://Phytophthora-id.org/) and Phytophthora DB (http://www.Phytophthoradb.org/), and local alignments.
Results: Phytophthora spp. were detected in soil taken from the root zone of plants in the restored areas (site 1-4). These were P. cambivora, P. europaea, and P. chlamydospora (Figure 2). No Phytophthora was detected in soil taken from dead and dying sword fern in the dieoff sites (sites 5-6). Of the three species detected in soil, only P. cambivora is associated with root disease and basal canker on its hosts (Table 2). Phytophthora chlamydospora is ubiquitous in soil and water and has occasionally been found to cause foliar blight on rhododendrons. Phytophthora europaea has been detected in soil and is weakly pathogenic to oak seedlings.
It is unlikely that a Phytophthora species is responsible for the sword fern dieoff at Seward Park, based on the results of our sampling so far. An experiment where healthy sword ferns are planted into soil from dieoff sites will help to determine whether a soilborne pathogen is present and causing the dieoff symptoms.
References:
Calmin G, Belbahri L, Lefort F. 2007. Direct PCR for DNA barcoding in the genera Phytophopthora and Pythium. Biotechnology Biotechnological Equipment 21:40-42.
Farr, D.F., & Rossman, A.Y. Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Retrieved June 13, 2017, from https://nt.ars-grin.gov/fungaldatabases/
Tamura K. and Nei M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10:512-526.
Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution30: 2725-2729.
Tidwell, T.E., Kosta, K.L., and Henderson, J.C. 1984. Root rot of western swordfern caused by Phytophthora cinnamomi in California. Pl. Dis. 68: 536. (2475)
USDA-APHIS 2010. Soil and container mix protocol. https://www.aphis.usda.gov/plant_health/plant_pest_info/pram/downloads/pdf_files/soil_protocol11-5-2010.pdf
White TJ, Bruns TD, Lee SB, Taylor, J.W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols — a Guide to Methods and Applications M.A. Innis, D.H. Gelfand, J.J. Sninsky, T.J. White, Editors , Academic Press, San Diego, CA, pp. 315–322.
