Soybean Disease Diagnostics Lab

Don Lee Author

05/16/2016 Added

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For more information about Oomycete Disease Diagnostics visit the website: https://ge.unl.edu/oomycete-disease-diagnostics/

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[00:00:08.298]My name is Kevin Korus,
[00:00:09.715]and I coordinate the Plant and Pest Diagnostic Clinic,
[00:00:12.295]which is located in Plant Sciences Hall, Room 448
[00:00:15.255]on east campus at the University of Nebraska, Lincoln.
[00:00:17.653]And the clinic is setup to process all different kinds
[00:00:20.554]of plant samples from anywhere
[00:00:22.133]in the continental United States.
[00:00:23.573]So we can do turf, grass, trees, landscape ornamentals,
[00:00:27.053]fruits, vegetables and row crops.
[00:00:29.013]Most of my plant samples are row crops
[00:00:30.653]that come from Nebraska including:
[00:00:31.931]wheat, corn and of course soybeans.
[00:00:35.000]Early on in the soybean cropping system,
[00:00:36.900]we do have many samples submitted.
[00:00:39.200]And a lot of the time the clients are interested
[00:00:41.640]in understanding whether or not their stand reduction
[00:00:44.399]was do to either pythium infection
[00:00:46.260]or a phytophthora infection.
[00:00:48.098]Identifying a disease based simply on symptom expression
[00:00:50.996]is just not feasible.
[00:00:52.376]It can be dangerous, you may get a misdiagnosis.
[00:00:55.496]So we encourage plants be sent to the diagnostic clinic.
[00:00:58.456]So that in the clinic we can use our tools
[00:01:00.575]to go in and specifically find those causal agents.
[00:01:04.254]Specifically find pythium or phytophthora,
[00:01:06.495]so we can say with confidence, yup this pathogen is here,
[00:01:09.455]you have to deal with it now in your growing conditions.
[00:01:13.095]When samples arrive to the clinic,
[00:01:14.554]the first thing I do is take a look at what the symptom
[00:01:16.675]distribution is like on the plant.
[00:01:19.255]In general, pythium will attack the roots.
[00:01:22.474]And the very lowest roots will be affected:
[00:01:26.054]will be brown, will be discolored and maybe you'll see
[00:01:29.515]root schlepping, when the root cortex pulls off,
[00:01:32.054]and the root steels left behind.
[00:01:34.554]Phytophthora on the other hand,
[00:01:36.275]even though that particular organism
[00:01:37.655]does also infect the roots,
[00:01:39.355]it works it's way up into the crown and lower stem of the
[00:01:41.454]plant a lot quicker.
[00:01:42.814]And we often see darkening and discoloration of the lower stem
[00:01:46.175]and crown when we have phytophthora.
[00:01:48.555]However, if these symptoms aren't really obvious
[00:01:50.595]on the plant, we may need to try to isolate
[00:01:53.795]from these plants and try to obtain a pure colony of that
[00:01:56.854]particular micro-organism causing the problem.
[00:02:00.234](water pouring)
[00:02:08.535]So the idea of using isolations for identification
[00:02:11.555]of plant pathogens is that
[00:02:13.435]if there is a plant pathogen present,
[00:02:15.674]that pathogenic organism is invading the plant tissue.
[00:02:19.055]It's inside those plant cells.
[00:02:20.815]And it's protected from the outside environment.
[00:02:23.434]What we want to do is try to get at that one organism
[00:02:26.475]and isolate it artificially,
[00:02:27.975]so it grows in culture here in the lab.
[00:02:31.412]What we don't want are all of the other organisms
[00:02:34.510]that are on and inside the plant not causing any disease,
[00:02:38.811]but are just kind of there hanging out.
[00:02:40.450]We don't want them isolated onto our plates
[00:02:42.530]because that's just going to confuse our diagnosis.
[00:02:44.910]So what we do is a surface sterilization
[00:02:47.685]with a 10% bleach
[00:02:49.925]and 70% alcohol solution
[00:02:53.262]that we mix 50/50.
[00:02:54.787]The idea is if there is a pathogen present,
[00:02:57.500]it's inside the plant and it's protected from that solution.
[00:03:00.584]So we'll take a large chunk,
[00:03:03.376]and we'll just place it
[00:03:07.827]right into this solution for 30 seconds.
[00:03:12.508]After 30 seconds,
[00:03:15.067]the root sample gets a 30 second wash
[00:03:18.287]in sterile distilled de-ionized water.
[00:03:22.906]After 30 seconds,
[00:03:26.666]the root sample is placed on a sterile dry paper towel.
[00:03:30.566]So, the next part is basically placing this plant material,
[00:03:33.746]which is now sterile, onto media.
[00:03:38.182]And the media types differ depending on whether we are doing
[00:03:41.123]a pythium or phytophthora isolation.
[00:03:44.383]For each isolation, there is a
[00:03:46.203]selected media for each specific organism.
[00:03:48.602]For pythium isolations, we have a pythium-selected media
[00:03:52.682]on which the root sample will be placed.
[00:03:55.482]We also place the root sample on water auger,
[00:03:58.483]so an auger that contains no nutrients.
[00:04:00.503]And the purpose of this auger is that fungi and water molds
[00:04:03.522]will sporulate when they run out of a food source.
[00:04:06.703]So if we put them on water auger containing zero sugars
[00:04:09.362]or zero nutrient sources,
[00:04:11.223]it'll encourage them to sporulate quicker.
[00:04:14.023]And as soon as they sporulate then we can identify them.
[00:04:16.661]So the sooner they sporulate,
[00:04:18.060]the sooner our turn around time is here in the lab.
[00:04:20.920]Since the plant sample is already sterile at this point,
[00:04:23.741]anything we do, any utensil that we use that touches
[00:04:28.041]this plant material, must also be sterile.
[00:04:35.536]So we don't need very much.
[00:04:37.976]What I do is I cut the tip of the root off.
[00:04:41.276]Then I'll cut three or four smaller sections of the root.
[00:04:50.795]And opening the plate as quickly as possible.
[00:04:59.316]After the sterile pieces of root material
[00:05:01.056]are placed on the media, we seal the plate with peri-film
[00:05:04.535]to make sure that no fungal spores enter in.
[00:05:08.836]It'll take anywhere between three to seven days
[00:05:11.176]for that organism to grow out and then sporulate.
[00:05:14.596]The sporulation is key.
[00:05:16.676]We use the spores to be able to identify and differentiate
[00:05:19.755]between different genuses and species of plant pathogens.
[00:05:24.135]Within a week, we should usually have spores on our plate.
[00:05:27.835]And what I can do is I can take that plate and flip it over
[00:05:30.895]and stick it on a microscope and look at it at 200 X.
[00:05:34.515]And I can see right through the media.
[00:05:36.295]If there are any spores that have developed,
[00:05:39.011]I can identify which species they belong to.
[00:05:42.990]So it's really important for a producer to get a diagnosis,
[00:05:45.591]a firm confident diagnosis,
[00:05:46.950]because you can't start the correct management plan
[00:05:50.730]until you have the proper diagnosis.
[00:05:53.170]And if you try to manage a disease that is not there,
[00:05:56.710]you can end up doing more harm.
[00:05:59.130]Or you could be spending money in areas
[00:06:01.150]where you don't need to be spending money.
[00:06:02.830]So again, you don't want to âŚ
[00:06:07.030]you don't want to get a false negative diagnosis,
[00:06:10.270]and treat for something that is not there, and you don't
[00:06:13.348]want to not treat for something that is there.
[00:06:15.868]Again, so it's just simply putting into place the
[00:06:20.048]strategies that will mitigate the problem
[00:06:22.148]that you actually have.
[00:06:23.568]And that starts with the proper diagnosis.

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Soybean Disease Diagnostics Lab

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