Poison Plate

Don Lee Author

05/23/2016 Added

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For more information please visit the Oomycete Disease Diagnostics web page: https://ge.unl.edu/oomycete-disease-diagnostics/

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[00:00:06.232]Don Lee here from
[00:00:07.950]the University of Nebraska-Lincoln,
[00:00:09.827]and I will concentrate on the work done
[00:00:12.892]by research pathologists to better
[00:00:15.958]understand fungicide response in oomycete.
[00:00:19.301]And again, I'll be concentrating on work coming out
[00:00:21.855]of Martin Chilvers' Lab at Michigan State University,
[00:00:25.478]and I think what's important here is,
[00:00:29.100]with this presentation, to give you a better idea
[00:00:32.815]of how pathologists actually work
[00:00:35.230]to obtain the data that agronomists
[00:00:38.434]and the farmers they support rely on
[00:00:40.896]when they make decisions about seed treatments,
[00:00:43.775]maybe seed treatments that have used in the past
[00:00:45.864]or seed treatments that are new on the marketplace,
[00:00:49.301]and then we can also see how researchers
[00:00:52.552]are coming up with methods that allow them
[00:00:55.710]to do their work more efficiently.
[00:00:57.893]I, again, wanna remind you that we are gonna take you
[00:01:02.537]through kind of the current status of seed treatments.
[00:01:05.323]I've got the extension resources link available
[00:01:10.013]in our learning environment for you to take a look at,
[00:01:13.357]but I do think it's real important to know
[00:01:15.958]what the choices are now and why knowing
[00:01:19.905]what those active ingredients are,
[00:01:23.109]what those fungicide products are,
[00:01:26.824]is important for agronomists and farmers.
[00:01:30.168]Instead what I'll do is focus on the research
[00:01:32.537]coming out of the pathologists' laboratory,
[00:01:38.695]where they're looking at fungicide sensitivity,
[00:01:41.427]and as we'll see, it's work that's still in progress,
[00:01:44.584]but we can see how they go about their work
[00:01:48.392]and how this could lead to better information in the future.
[00:01:53.547]So what you need to understand is what they mean
[00:01:58.423]by this poison plate method.
[00:02:00.745]They refer to it as their gold standard,
[00:02:02.789]and so when Dr. Chilvers draws yeast circles here,
[00:02:07.247]every circle represents a Petri dish
[00:02:09.522]that has a growing environment media
[00:02:13.238]that's conducive to the growth of oomycete mycelia,
[00:02:19.043]so the more those mycelia grow and spread,
[00:02:24.198]you can see a growing diameter on your Petri dish.
[00:02:29.678]Okay, takes a few days to conduct this.
[00:02:31.582]But then if you want to understand
[00:02:33.625]how their growth occurs in response
[00:02:36.597]to a certain concentration of fungicide,
[00:02:40.545]you can put various doses of a fungicide
[00:02:44.631]in the media and then look at growth rate
[00:02:49.182]over the same time period, compared to the control
[00:02:52.891]that has no fungicide.
[00:02:54.841]And then the measurement they're looking for is the EC50,
[00:02:58.324]the concentration of the fungicide
[00:03:00.275]that reduces the colony diameter by half,
[00:03:02.922]so if we measure that colony diameter,
[00:03:05.198]about half of it would be maybe somewhere
[00:03:08.031]between these two plates, that's where
[00:03:10.399]the EC50 would of this particular fungicide.
[00:03:14.253]So it's the gold standard. This is what it looks like
[00:03:17.040]on real plates. Martin added in the circles here
[00:03:20.277]so we could see the extent of the mycelial growth.
[00:03:23.202]Two different isolates, two different oomycetes.
[00:03:26.592]Which one has the lowest EC50?
[00:03:32.909]The Pythium or the Phytophthara.
[00:03:35.973]Yeah, you can see by the diameters,
[00:03:38.017]the Phytohphthara grows at a slower rate,
[00:03:42.614]but it's EC50 is occurring
[00:03:45.679]at a much more dramatic rate than the Pythium.
[00:03:50.973]Okay, so differential response to this particular fungicide,
[00:03:55.478]and you can see that response from the poison plate method.
[00:03:59.565]The challenge with the poison plate method
[00:04:01.562]is just the magnitude of the nature of the studies
[00:04:04.348]when you're looking at hundreds of different isolates
[00:04:07.924]that are potentially found in a farmer's field.
[00:04:12.661]You have to pour a lot of plates
[00:04:14.054]that have these various concentrations,
[00:04:16.887]and because some isolates will respond
[00:04:22.181]in a different way than others,
[00:04:25.757]they'll grow maybe at the same rate in the control plate
[00:04:30.819]but then have a differential response in their growth rate
[00:04:34.209]to increasing concentrations of the fungicide.
[00:04:38.760]And when you have a lot of isolates,
[00:04:40.339]and if you have new experimental fungicides
[00:04:44.008]that you're testing, these poison plate assays
[00:04:46.516]can be pretty labor intensive
[00:04:48.884]as well as supply intensive, resource intensive.
[00:04:53.296]But if you do a good job of conducting these studies,
[00:04:55.943]you get very important information,
[00:04:58.172]so for example, we can see that this particular fungicide
[00:05:00.959]might not have the same response to every isolate
[00:05:04.534]that would be in a farmer's field,
[00:05:07.135]and so if this is an experimental fungicide,
[00:05:09.271]there may be some decisions about
[00:05:13.404]whether or not this should be continued to be tested
[00:05:15.819]as a potential product that farmers could use
[00:05:21.206]to protect their seed health
[00:05:24.875]in the early stages of the growing season.
[00:05:27.568]Poison plate method is the gold standard,
[00:05:29.844]but it's labor intensive and costly.
[00:05:33.280]So, the Chilvers lab has investigated
[00:05:36.299]how new technologies and new resources
[00:05:38.761]might be put together to come up
[00:05:41.593]with an alternative to the poison plate method,
[00:05:44.380]and so this involves some sophistication
[00:05:47.212]with pipetting, the use of these microtiter plates.
[00:05:50.649]They have a little well that a liquid can be placed in
[00:05:54.272]and you can have mycelia growth occurring in that well
[00:05:57.569]and then you can take advantage of machines
[00:06:00.401]that do a good job of reading how well light travels
[00:06:03.838]through these microtiter plates,
[00:06:06.207]and the more mycelia growth,
[00:06:08.575]the different rate at which light
[00:06:13.219]will pass through those chambers.
[00:06:15.402]Okay, so here is the outline that the Chilvers lab shared
[00:06:19.210]to show their new method, so let me take you through it.
[00:06:23.343]You can grow mycelia from your different isolates
[00:06:27.104]on this agar media for a few days,
[00:06:30.402]and pull out some constant amount of mycelia
[00:06:34.296]from plugs that are then macerated
[00:06:37.547]with a syringe in the agar, and then they're vortexed
[00:06:41.122]into liquid and then you can create a reservoir
[00:06:47.067]of the mycelia from this particular isolate, okay.
[00:06:52.593]And then what you can do is set up these microtiter plates
[00:06:57.469]so that you have a control and then you have
[00:07:00.023]increasing concentrations of your fungicide.
[00:07:03.460]And in this case, they're in a liquid media,
[00:07:06.664]not in a solid media, and you can then let them
[00:07:13.119]grow for 24 hours and they're rate of growth
[00:07:16.835]will be related to the optical density
[00:07:19.482]that you get by reading the plates in this machine,
[00:07:22.733]and the specific wavelength.
[00:07:24.962]Alright, so, that's the microtiter method,
[00:07:30.302]the plate reading method,
[00:07:32.764]and so in order to determine if this new method is valid--
[00:07:36.804]does it give you the same results
[00:07:39.126]as the gold standard, the poison plate method?
[00:07:41.587]You could conduct a study where you take the same isolate,
[00:07:45.652]and you test its EC50 with both methods.
[00:07:50.523]I'm gonna share with you a couple of different
[00:07:52.335]potential outcomes from this experiment,
[00:07:55.446]and then we'll see the outcome
[00:07:57.071]that they really got in the Chilvers lab.
[00:07:59.672]Okay, so you could have an outcome that looks like this,
[00:08:03.527]where if you get a low reading
[00:08:05.431]from the optical density method,
[00:08:07.427]you get a low method from the poison plate method.
[00:08:10.400]If you get a higher reading from the optical density reading
[00:08:15.926]you also get a higher reading on the poison plate method.
[00:08:20.744]Occasionally, you might see an outlier
[00:08:22.700]that doesn't fit that, but generally,
[00:08:24.790]you have a strong correlation between
[00:08:26.833]the results of one method and the results of another method.
[00:08:30.595]That's one potential outcome.
[00:08:32.174]What would be another outcome
[00:08:33.985]that you could potentially see?
[00:08:35.610]Well, you might see that if you conduct this experiment,
[00:08:39.000]your data is scattered, alright, your data is scattered.
[00:08:44.898]There doesn't seem to be a strong correlation.
[00:08:47.280]You might get a high reading with one method
[00:08:48.858]and a low reading on the other method,
[00:08:53.085]and so the lack of correlation gives you less confidence
[00:09:03.533]that the new method would give you the same result
[00:09:06.599]as the gold standard, the currently accepted method.
[00:09:12.079]So what were the results that the Chilvers lab obtained?
[00:09:14.865]I accidentally kinda gave you a preview to this.
[00:09:17.419]They saw some scattering in their data.
[00:09:21.015]Didn't all follow along the same line.
[00:09:24.730]But when they look at the statistical correlation,
[00:09:27.145]there is a high correlation.
[00:09:28.677]So that means that the results from
[00:09:30.675]the more efficient optical density method
[00:09:34.529]were consistent with the results
[00:09:37.083]that they got from the poison plate method.
[00:09:40.613]Alright, so that gives them confidence
[00:09:43.167]in this optical density method.
[00:09:45.767]Might be very useful in conducting studies
[00:09:48.646]where you're trying to compare the efficacy
[00:09:50.922]of two different seed treatments, two different fungicides,
[00:09:55.612]so you can see that the one fungicide in the dark blue
[00:10:00.746]has a very high optic EC50,
[00:10:09.531]especially across some of the species,
[00:10:12.967]in contrast to this other fungicide, which tends to show
[00:10:16.868]a much lower EC50 across all these different species,
[00:10:23.463]and so with the diversity of species
[00:10:25.971]that are in farmers' fields, one may be a better choice
[00:10:29.082]than the other in order to prevent oomycete disease.
[00:10:34.562]So being able to use this optical density method
[00:10:37.813]allows them to conduct this research on a grander scale,
[00:10:43.571]which is gonna be very important
[00:10:45.243]if they wanna test them across the wide variety of species,
[00:10:49.005]maybe to look at different temperatures,
[00:10:51.327]and be able to repeat these studies
[00:10:56.342]on experimental or potentially new fungicide treatments
[00:11:00.104]that could be good choices for farmers in the future.
[00:11:03.448]So the results look positive so far.
[00:11:06.884]More research has to be done to increase
[00:11:09.857]their confidence in this new method.
[00:11:12.550]So we can see how pathologists go about their work,
[00:11:15.893]how they obtain the data, and how they're looking
[00:11:18.680]at doing a better job of this.
[00:11:20.445]And then again, when we look at field such as this,
[00:11:23.185]where the soybean farmer has the potential
[00:11:26.157]to have a good year of soybean production
[00:11:28.525]and they'd like to have that uniformly across their field,
[00:11:33.355]they might be able to have better choices, new choices
[00:11:36.931]that they could take advantage of in the future,
[00:11:39.067]and hopefully this research contributes
[00:11:41.017]to those future choices.

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Poison Plate

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