Environmental Sequencing?

Culturing and PCR have not yet revealed pathogens which may be responsible for the sword fern die-off.  Here are some readings and references, gathered unsystematically from the recent literature, about the use of next-generation, environmental sequencing and microarrays for identifying pathogens.

1. Metagenomics (wikipedia)
2. Putative pathogenicity genes of Phytophthora cinnamomi identified via RNA-Seq analysis of pre-infection structures
3. Moleculo long-read sequencing facilitates assembly and genomic binning from complex soil metagenomes
4. GeoChip as a metagenomics tool to analyze the microbial gene diversity along an elevation gradient
5. The PathoChip, a functional gene array for assessing pathogenic properties of diverse microbial communities

Die-off at Seattle Park Cheasty Mountain View

Tim, Al and I followed up on Al's initial report of sword fern die-off at Cheasty Mt.View, a few dozen yards west of the intersection of Columbian Way and Mountain View Drive.

The area is second growth forest, mostly big leaf maple, with a mixed sword fern, Indian Plum, salal and invasive understory.

We did an informal survey of a mixed flat and steeply sloping area, about 135 feet east-west by 100 feet north-south.  We placed 110 orange marker flags at each unambiguously dead fern; closely clumped dead crowns were treated as a single fern.  We excluded any fern showing any trace of green. This yields a concentration of  ~120 sq.ft per fern - which is consistent with a second growth, mixed understory rich in ferns.  The understory of this site is decidedly not dominated by ferns in the manner that the Seward, Suquamish and Upper Luther Burbank sites are.

Tim, Al and I then each separately estimated the ratio of dead/live ferns across the  135 x 100 foot region.  This was exceedingly informal.  Our average estimate was about 50%.  We did not distinguish a third group:  affected ferns still showing some green.  That group was lumped in with the healthy ferns.

This site does not meet the six criteria - which were inferred from die-off sites in which the sword ferns been the dominant - indeed, nearly exclusive - understory plant.

We left ten short orange marker flags in ferns with medium-to-good health.  One of Tim's natural history students will do repeat photography of these ten ferns over the next two months.

Suquamish (Kitsap) Die-Off: stats and video

Site walk-through

Yesterday (Saturday, April 8th) I returned to private property, on the Kitsap Peninsula, near the Suquamish Reservation.   The owner first noticed extensive die-off in 2010, making this the first report of the phenomenon.

Some details:

1. west-facing slope, dominated by large red cedars, from 2'-4' dbh.  Some big leaf maple.
2. 140 dead crowns in 2300 sq ft: crown density about 16 sq ft.
3. two surviving ferns for density comparison: large at 36 sq ft, small at 9 sq ft
4. no doug firs in the 2300 sq ft
5. good regeneration of other species, mostly Indian Plum, some bracken fern
6. crowns are marked with orange flags (otherwise hard to see in the video among small new Indian plum)

Table of Contents

Introduction

 Left: looking east towards Ground Zero, c. 2010

Right:  same scene, 2016

Two Informally-Validated Die-Off Sites beyond Seward Park

The Die-Off Jumps the sqebeqsed trail

New Sample Collection with Marianne Elliott (WSU Puyallup)

Field Criteria for Identifying a Die-Off Site

The Ntirogen Deficiency Hypothesis

Individual sword ferns apparently live for centuries

Narrative Summary (including comments from NY Botanical Garden’s fern curator, 

  and world expert genus taxonomist  from the University of Vermont)

January 10 2016:  more die-off found in the northwest corner of Seward Park (with video)

December 27 2016: die-off on Mercer Island's Upper Luther Burbank Park  (with video)

Paul Talbert: Comments on PCR Results 

WSU Puyallup Phytophthora PCR Results 

Novermber 13: a presentation to the Washington Native Plant Society, by Dr. Tim Billo and undergraduates Kramer Canup and Tristan O'Mara 

September 22: Grant Proposal to the Bullitt Foundation 

Informal final report from UW’s Tim Billo and his two undergraduate students
to the Seattle Parks Field Committee (June 2nd, 2016)

Lab report from Dr. Marianne Elliott, WSU

LSU Professor Valverde, plant pathologist, 2008 paper on “new virus causing disease in [a related] fern”

Site visit by WSU pathologists, and the “introduced phytophthora” thesis

PCR and Sequencing for Pathogen Detection (July 25th, 2016)

Two Informally-Validated Die-Off Sites Beyond Seward Park

On private property on the Kitsap Peninsula, near Sandy Hook, the owner has watched their several acres of sword-fern dominated second growth forest die off.  This beganin 2010, and is thus our earliest report so far.  Sword fern root and soil samples are under analysis at WSU Puyallup.   This site meets the proposed six criteria by which a die-off site is identified.

The die-off occupies a gentle west-facting slope with a marked (possibly relevant) absence of doug firs.  Cedars and maples dominate.  The area is largely open beneath these moderately spaced second-growth trees.  Regeneration is taking place, including (at least) the native Indian Plum.

The Upper Luther Burbank Park site is smaller.  Its status as a die-off site is, I believe, contested by Mercer Island Parks staff.    This legitimate, and currently unresolved difference of opinion partly inspired my six proposed die-off criteria.  This site meets all criteria except number six: that the die-off area has been assayed in successive years, and is growing radially.     Annual monitoring of the Mercer Island site will resolve this.

The Die-off Jumps the sqebeqsed trail

Over the last three years our attention, and our monitoring efforts, have been mainly upon the southeast sector of the Magnificent Forest, south of the Hatchery Trail, east of the sqebeqsed trail.

On an evening walk in the woods on Wednesday (5 April 2017) I was surprised and alarmed to see many instances of new brown sword fern crown stumps within a few hundred feet of ground zero, on the west side of the sqebeqsed, on the Andrews Bay Trail.  See map below.

This new discovery, along with our current pessimism about identifying the cause of the die-off, and the two informally validated new sites (Suquamish Sandy Point on the Kitsap Peninsula, Upper Luther Burbank Park on Mercer Island), triggered a state change in my assessment.

Which is that the die-off will likely continue to propagate through the Magnificent Forest in semi-exponential growth - that is, faster than linear, but not so fast as annual doubling.  It is likely therefore, that within ten years all of the sword ferns will be dead or dying.  Furthermore, and for reasons unknown, regeneration is non-existent in all of our die-off areas.  Ground zero has been open for colonization for three years; not one new plant has appeared. (See reference below for possible insight into this "regen problem" based upon 2012-13 research at Lincoln Park.)

There exists, therefore, a grim possibliity:  that a full half of the understory will be destroyed without replacement, disrupting the structure and likely the function of the old-growth forest with unknown but worrisome consequences.

Note that the two other informally validated die-off sites, on Mercer and the Kitsap, ARE regenerating.   Seward is an anomaly in this regard, perhaps due to coarse woody debris removal in the 1960s, and the absence of any of the well-known old forest nitrogen sources.

Seed limitation and lack of downed wood, not invasive species, threaten conifer regeneration in an urban forest  Ailene K. Ettinger,  Benjamin R. Lee, Sarah Montgomery

New sample collection with Marianne Elliott

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.

Marianne's Progress Report:  6/14/2017

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.