Summary by Tianna DuPont, WSU Extension, Scott Harper, WSU Pathology. May 28, 2021. Updated Aug 23, 2021.
As you sample and test for X-disease and Little cherry disease here are a few things to keep in mind as you read your results.
Your results are only as good as the sample you send in. Make sure to sample four five-inch cuttings including leaves, (fruit okay) and fruit stems from symptomatic limbs (on symptomatic trees) or from each leader (non-symptomatic trees). Don’t let your samples get too hot in the field, the truck or in the mail. Sample when symptoms are visible (generally starting a week before harvest) until the concentration starts to drop off in the tree in the fall. For sampling instructions and videos and symptoms click.
Type of test
WSU/OSU recommend testing be done using real-time qPCR (quantitative Polymerase Chain Reaction) or RT-qPCR (Reverse-Transcription-qPCR) for the virus using optimized, validated methods. Much like standard PCR, the reactions involve amplification of a fragment of DNA through sequential heating and cooling steps with enzymes that essentially make more copies of the target DNA. Quantitative PCR is different in that rather than waiting until the end of the reaction and analyzing the product on a gel, it uses a fluorescent probe or dye that reports the signal in ‘real-time’ as it is amplified, which is faster and more sensitive. This also allows you to calculate how much DNA you started with in your sample, because a sample with a high concentration of starting DNA will produce a signal faster than one with less.
To ensure that all participating labs are using the tests correctly, and producing accurate results, WSU has been providing a series of proficiency panels. For a listing of labs click here. All of the participating labs are using the same methods validated by the WSU Harper lab (listed at link).
Why do I sometimes get variable results?
When a tree has been infected for some time the number of virus particles or phytoplasma pathogen cells in the tree is high. However, if the tree was recently infected there may be only a small number of pathogen cells/particles in the tree. Your probability of getting a piece of tissue with enough pathogen cells to detect can be low when the tree is newly infected. A sample from one limb may be positive and another limb may be negative. Even within the same sample one extraction in the lab may happen to get pathogen cells and another not get any if the original number of cells in the plant is very low.
What is the Ct value?
Some labs may report a Ct or Cq value. This is the number of times the machine amplified (multiplied) the DNA before detecting the pathogen in the sample. The higher the Ct number the lower the concentration of the pathogen there was in the plant. Ct values of 20-30 indicate a relatively HIGH pathogen concentration in the plant. Ct values of 37-40 indicate a very LOW concentration of the pathogen in the plant. When concentration in the plant is very low (high Ct value) it is normal that some samples may turn out positive and others negative since it is a matter of chance that the sampler and the lab happen to get a piece of the plant with pathogen in it.
That being said with any test, errors can happen during extraction or processing and if you see very obvious symptoms but results are negative it is a good idea to re-sample and/or ask the lab to re-run the test.
Can my Ct value change over the season?
The concentration of the pathogen in the tree changes over the course of the season. In the spring the concentration can be very low resulting in no detection or a high Ct value (eg 38). Over the season the concentration in the plant tissue increases and the resulting Ct value may be lower (eg 27).
Aggressive tree removal is critical to slow the spread of the pathogen. For blocks where either X-disease or Little cherry disease have been previously confirmed prompt removal without testing is recommended when symptoms are clear. When symptoms are unclear or the disease has not been previously confirmed sample, test and promptly remove diseased trees. Generally, test results are a simple positive or negative. Consider the specifics when you have unusual results.
Tianna Dupont, WSU Extension (509) 293-8758 email@example.com
Scott Harper, Department of Plant Pathology, Washington State University (509) 786-9230 firstname.lastname@example.org
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