Presentation to the Washington Native Plant Society

November 3rd, 2016

Presentation to Washington Native Plants Society

Some Comments on PCR Results from Paul Talbert

November 18th and 20th, 2016

Thanks, Katie.

I guess I am relieved we don't find evidence for Phytophtora infection, but that leaves us with no good hypothesis.

I'll look forward to hearing about the bait experiments. Meanwhile, I guess we should try to think of other hypotheses.
 

So thinking out loud here, it looks like your positive control has about 16,000 times as much Phytophthora DNA as the healthy soil, which has about 4x as much as the affected soil. The latter difference may be within sampling variability/error. So there is no evidence that a Phytophthora is causing our problem, but we do apparently have Phytophthoras in our soils, since nothing came up in the water.

Assuming we get nothing of interest again in the bait experiment, would there be any point in repeating the experiment on growing roots in the spring when the fiddleheads are coming out? I don't want to chase phantoms or beat a dead horsetail (that was a fern ally joke), just wondering if we could be missing anything. You don't have to answer now; we have till spring to consider.

Katie Coats and Marianne Elliott of the WSU Plant Diagnostics Lab in Puyallup: a report after PCR assay of Phytophthora at the genus level

(received 18 November 2016)

(In early November, I collected samples from a healthy fern - fine roots and nearby soil - off the windfall trail, and from an affected surviving fern just west of ground zero, and delivered them to Katie in her lab - Paul Shannon)
 
I have qPCR results on the sword fern samples but they're not conclusive.

I extracted DNA from the following samples:

1. "healthy soil a" = 4 grams of soil from 12" away from healthy plant
2.  "healthy soil b" = 4 grams of soil from 24" away from healthy plant
3.  "healthy plant" = roots, surface sterilized in 10% bleach for 1 minute then rinsed twice in sterile water to assure qPCR is detecting infection inside the roots, photo attached
4.  "affected soil" = 4 grams of soil from near affected plant
5.  "affected plant a" = roots, surface sterilized in 10% bleach for 1 minute then rinsed twice in sterile water, photo attached
6.  "affected plant b" = split from sample 5, run as a replicate

The DNA samples were run in duplicate through the general Phytophthora (5.8S) qPCR assay along with a positive control (DNA from a pure culture of Phytophthora) and a negative control (water). All samples were multiplexed with an amplification control that checks for evidence of PCR inhibition, something that’s common in root and soil samples.

Results are shown in the bar graph below… it’s crude… I don’t usually use the Ct values, but I think it’s a quick way to get the results across to you. The lower the Ct value, the more target DNA is present… so it makes sense that the pure DNA sample has a Ct around 16.  The qPCR runs up to 42 cycles, but data above 40 cycles is dubious. If no phytophthora is present, then there will be no Ct value at all, which was the case with the water control. Theoretically the DNA doubles in each cycle, so a Ct value one more than another represents twice as much target DNA.

he bottom line is that the healthy soil and plant generally had lower Ct values (more Phytophthora DNA) than the affected soil and plant. I don’t have a soil sample from another location outside the park to give us an idea of how normal these levels of phytophthora are, but they’re counter what we expected...  One thing to keep in mind is that this test lumps all phytophthoras together, so it remains possible that a causal phytophthora is in higher concentration in the affected plant even though the total amount of phytophthora is lower.

Therefore, we will proceed with another strategy referred to as baiting. We’ll add some rhododendron leaves as baits to the individual plant and soil samples along with water… and then plate in culture medium to see if/how much/which phytophthoras may be present. That will take a couple weeks from start to finish… so more wait time.

I’ll send you another update once we know the timeline of those results; ballpark of early December.

I’m including Marianne in case she has other comments… and let me know if you have any questions.