Dr. Marianne Elliott's Report (12/3/2015)

I have attached the sampling procedure and list of samples we collected. We did not find any Phytophthora, but I am not ready to rule it out as the cause of the die-off. It could be a species that is slow growing and difficult to culture, so we missed it. Or it was replaced by something else in dead material. Or this was the wrong time of the year to sample. I think the students who are working on this should keep an eye out for it.

There were a few Pythiums and other similar organisms that we see commonly in soil and roots. We didn't find anything associated with the dying sites vs the healthy sites, or with the dead cedar transplants. I have been asking various people I know and nobody has seen it in their travels, but now they are aware of the situation. I will let you know if anything turns up.

--Marianne

Sample Processing Protocol (Sword fern dieoff samples collected at Seward Park 11/10/15)

Sample #	Description	Treatment
1	5 x 5 plot, healthy, symptomatic fern foliage	Surface sterilize, plate 5 segments on PARPHV8 (3 plates)
2	5 x 5 plot, healthy, crown from healthy fern plant	Surface sterilize 10 necrotic root segments per plate (PARPHV8), dissect crown and look for brown staining symptoms (3 plates). If present, surface sterilize and plate 5 segments on PARPHV8 (1 plate).
3	5 x 5 plot, healthy, soil from base of 3 healthy ferns, upper 5 cm depth + litter & roots	Bait in 1 L bottles. Use rhododendron leaves and healthy sword fern leaves. Place one intact rhody leaf and one sword fern frond in each of 3 bottles containing 100 g soil and 500 ml water.
4	Ground zero, foliage and fronds from dying ferns	Surface sterilize, plate 5 segments on PARPHV8 (3 plates)
5	Ground zero, crown and roots from dead ferns	Surface sterilize 10 necrotic root segments per plate (PARPHV8), dissect crown and look for brown staining symptoms (3 plates). If present, surface sterilize and plate 5 segments on PARPHV8 (1 plate).
6	Ground zero, soil from base of 3 dead ferns, upper 5 cm depth + litter & roots	Bait in 1 L bottles. Use rhododendron leaves and healthy sword fern leaves. Place one intact rhody leaf and one sword fern frond in each of 3 bottles containing 100 g soil and 500 ml water.
7	Ground zero, soil from base of 3 dead, planted cedars	Bait in 1 L bottles. Use rhododendron leaves and healthy sword fern leaves. Place one intact rhody leaf and one sword fern frond in each of 3 bottles containing 100 g soil and 500 ml water.
8	Symptomatic foliage from other hosts along trail by Ground zero and road	Surface sterilize, plate 5 segments on PARPHV8 (3 plates).

Baiting protocol for soils

Label 1L bottles with sample and rep numbers. Use rhododendron leaves and healthy sword fern leaves. Place one intact rhody leaf and one sword fern frond in each of 3 bottles containing 100 g soil and 500 ml water. Cap tightly and incubate bottles on their sides for 48-72h.

After incubation, remove baits from bottles and rinse with water, then blot dry. If asymptomatic incubate an additional 3-7 days in ziplock bags containing moist paper towels. Plate symptomatic areas of foliage (5 per plate) on PARPHV8. Use two plates for each bait type per bottle.

All samples

Check for Phytophthora colonies on all plates after 2-5 days and isolate onto small PARP plates for identification.