Webinar #5 - How do I determine whether water is clean or contaminated? (February 2014)

By: Gary Moorman

Webinar Recording

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►How do we determine water is clean or contaminated?  
(► = PowerPoint image)

I'm Gary Moorman, a professor of plant pathology at Penn State University.

►This is one of a series of webinars organized by the research team on the Specialty Crop Research Initiative Project titled, Integrated Management of Zoosporic Pathogens and Irrigation Water Quality for a Sustainable Green Industry. The project is funded by the USDA National Institute for Food and Agriculture and the research team is from several universities and the USDA-ARS as you can see from the various logos displayed here.

►These people are the research team leaders

We would like to thank the panel of growers, the Society of American Florists, and the American Nursery and Landscape Association for their advice and assistance on this project. They are key partners in this project.

A major goal of this project is to gain a better understanding of the biology of plant pathogens in irrigation water and educate people about the topic. Based on this better understanding, we hope to be able to mitigate these pathogens.

Each of the webinars in this series has been recorded.

►In previous webinars, I discussed what plant pathogens have been found in irrigation water.
Then in another webinar I detailed how those plant pathogens might get into irrigation water.

►Today the question is…
How do we determine whether irrigation water is clean or contaminated with a plant pathogen?

That is a crucial question to be answered BEFORE any action is even considered.
In my experience, this question is only posed when crop losses are occurring or have occurred at some time.

►The first question to be answered is,
What plant pathogen is causing the crop losses?
We learned in an earlier webinar that plant pathogens from almost every major group of living organisms have been found in water.
But there are many different plant pathogens that can infect a crop.
Some plant pathogens are NOT likely to be waterborne and other sources should be examined first.
However, certain other plant pathogens are known to be associated with water or sediment, etc.

If you are losing plants because of one of those plant pathogens, then it is worth considering irrigation water as a possible source.
We might be looking for a needle in a haystack and it would be helpful to know what the needle looks like.
We can't look for every plant pathogen. There just isn't enough time or resources for anyone to test for everything.

Bottom line = Determine which plant pathogen is of most concern.
Any plant disease clinic can help but determining which plant pathogen is causing crop losses.

For the rest of the webinar today, let's assume that we know the identity of the plant pathogen causing crop losses and we suspect that the pathogen could be dispersed via irrigation water.

We want to determine whether or not that particular pathogen is in the water.

►Who is going to determine whether or not the water is contaminated?
That is a huge question. To my knowledge, there is no commercial laboratory that routinely tests water for plant pathogens.
Water testing labs routinely test for alkalinity, hardness, pH, radon, coliform bacteria that may cause human diseases but not plant pathogens.
Dr. Paul Fisher at the University of Florida has kindly granted permission for us to make a chart available of information he gathered on resources for testing water.
We have posted it for you to download.
It lists possible labs where a test for a plant pathogen might be run and also the source of test kits that I will be referring to.

1. You need to find a lab willing to test the water for you for plant pathogens, OR
2. You need to gather the materials to test it yourself.

►A first stop is the plant disease clinic in your state. They may be willing to help with the testing.
If your state university has a college of agriculture, it probably has a plant disease clinic or a nearby state has one.
The clinician may be willing to run the test or help you find a clinic or a researcher who can do the testing.

The table of information collated by Dr. Paul Fisher at the University of Florida provides some information on lab testing facilities.

Note on Dr. Fisher's chart that the University of Massachusetts and University of Florida labs may be willing to test samples. But it is important to contact them first to verify what they are willing to do it.

At this time, I am willing to test water samples from growers in Pennsylvania and from growers participating in our Specialty Crops Research Initiative project for Pythium.
Dr. Hong is willing to test water samples from growers in Virginia and from project participants for Phytophthora.

It is possible that if you are outside our states and not involved in the project that Chuan and I could be convinced to test your water for Pythium or Phytophthora. For sure, we may be able to help you find other researchers who may be willing to look for plant pathogens.

I would like to go through what would be done to detect Pythium or Phytophthora in water. As I do so, I will point out what a farmer or a clinician could do... someone who doesn't do this for a living.

►So, you have a diagnosis and know that either Pythium or Phytophthora is involved.
You want to know if your water is the source of the pathogen.

►Before doing any sampling, contact the person who will be doing the tests to make sure that person knows the sample is coming, when it is coming, and is given the opportunity to suggest how to collect a good sample.

►Sampling the water
The method used has to be appropriate for the plant pathogen sought (the target organism).

►There are two main ways of sampling for Pythium and Phytophthora

  1. Grab sample = collect some water for processing at a particular place at a particular time.
  2. Baiting = put organic matter of some sort in the water for a specified length of time to allow the target organism to colonize it. Tests are then run on the colonized bait.

►Grab Sample
At a particular time, you collect a water sample. Usually a sample is collected at each major point in the irrigation system. Many farms have more than one source of water, more than one holding impoundment, and more than one distribution system. Samples should be collected at key points.

►Collect samples during the growing season. At outdoor production facilities, do not collect samples in the middle of the winter. In greenhouses if there is little or no production going on in the summer, do not collect samples in the summer.

If the farm or greenhouse is recycling irrigation cycle, collect a sample at a central point as the water is coming off the crop.
Another place to consider is a sample near the intake point before it enters a pump for irrigation.

2 liters per point should be plenty for each sampling site.
Don’t’ mix water from different locations. Put them in different bottles.
I suggest NOT collecting at the very bottom of the water column.

Use a clean container. It can be a clean milk jug or soda bottle if Pythium or Phytophthora are the targets.
If bacteria or viruses are the targets, you want a new container that never had anything in it previously.

You can attach a rope to a bucket or other wide-mouth container, hang onto the end of the rope and throw the container into the water. Then transfer that water into your milk jug or bottle.

There are special pieces of equipment that can be purchased that remain closed until you place them at a specific depth that then can be opened. However, I don't believe those are necessary. Collecting from near the water surface should be sufficient.

Keep the sample out of direct sunlight. That means, put them in a cardboard box or a cooler immediately if at all possible. If you put cold paks in the box or cooler, it is best to not place them in direct contact with the sample bottle. Put some pieces of cardboard or bubble wrap between them. I put cold paks in the bottom of the cooler, then put a piece of cardboard over them and place the sample bottles on the cardboard.

Ship them to the person who will process them as soon as possible. If you use UPS, FedX or the US Mail, you must be absolutely certain that the containers will not leak. If they leak, they will not be delivered.

Seal the bottles well. Tape the caps on. Label each container well with a waterproof marker. Or if you use some type of tag, make sure to use a waterproof marker and a tag that will not fall apart if it gets wet. Put individual bottles in a plastic bag and seal the bags well. Place those in a sturdy cardboard box along with absorbent material to cushion and protect the bottles... and soak up any small leaks.

In a separate plastic bag, put in a note describing what is in each of the bottles, where the sample was collected, when it was collected, etc. Seal that bag well. Put that in the box or attach it securely to the outside of the box.

Ship the samples as soon as possible or hold them in a refrigerator until they can be shipped.

Make sure you ship them overnight express, or on a day that the package will be delivered promptly and not left over a weekend in a post office or receiving office.

Most universities do not distribute mail on Saturday or Sunday or holidays. For example, if a package arrives at Penn State Friday afternoon, it will not be delivered to me until Monday.

If you are going to process the sample, collect the sample the day you are going to process it or store it in the refrigerator.

Once the sampling is done and the samples have gotten to the person who will be processing them, now the sample has to be processed.

Usually, plant pathogens in the water is there in low concentrations.
It is not likely that if we take some of the water right out of the bottle and put it on agar to isolate the pathogen or run some other test on the raw sample that the pathogen will be found.
It is best to concentrate the pathogen in some way.

►One of the easiest ways is to pass the water through a filter paper that has a pore size that will retain the plant pathogen. For Pythium or Phytophthora, that’s 5 µm pore size. The water passes through but Pythium or Phytophthora are retained on the filter.

►Dr. Hong and my lab, we put the filter paper in a funnel that holds it attached to a vacuum pump and pull a low vacuum to speed the process. Sometimes an entire liter will pass through easily. Sometimes we need to use several pieces of filter paper.

►We then invert that filter paper on an agar that contains antibiotics to inhibit almost everything except Pythium or Phytophthora.

After 24 hr of incubation, we remove the filter paper and then check the plates each day for a couple days for colonies that look like Pythium or Phytophthora.
Once found, then we determine which species of Pythium or Phytophthora was isolated.

►Another approach would be to use one of the test kits available through Agdia, Inc. or Neogen. See Dr. Fisher's chart. You purchase one kit specifically for Pythium and a separate kit specifically for Phytophthora.
Anyone can use these kits. They are very easy to use and relatively inexpensive.

►You get all the materials required. This is an example of an Agdia kit.

►With the kit, you put the sample in a little bag of buffer that comes with the kit, grind it up, and then put in the dipstick for the Agdia test or

►apply some of the liquid to the test strip for the Neogen.

If you have filtered the water, then put part of the filter in the bag of buffer. That is your sample.
In fact, you could cut the paper in half and test one half for Pythium and the other half for Phytophthora.

 In 10 -20 minutes, you should see at least one line become visible, the quality control line. That line indicates that the test strip is working. If the quality control line does not appear, that detector is defective. In my experience, that is very rare.

If the quality control line and a second line appears, then the test is positive for Pythium or Phytophthora

►If the quality control line appears as it should but no second line appears, what does that mean?
It means that the target pathogen was not in the sample.
It could mean that the pathogen is not in the body of water where the sample was collected.
However, it is possible that the target pathogen is in the body of water but you just didn’t happen to collect any when you took the sample.
You might want to take additional samples during the season to verify that the pathogen is not detected.

What does a positive test tell you?
In this example, it is probable that Pythium or Phytophthora was in the sample.
That's good to know BUT, you need more information.

►The kits are for the genus of Pythium or the genus Phytophthora.
There are over 200 different  species of Pythium and may be that many species of Phytophthora .
A kit does not tell you which species of Pythium or species of Phytophthora is present.

In the case of Pythium, many different species have been found in water.
Most Pythium species are not plant pathogens and some are very weak plant pathogens.
Relatively few species are serious plant pathogens.
The test kit will give you a positive result regardless of which species is present.

In the case of Phytophthora, most species are plant pathogens but some are weakly plant pathogenic.
The kit only tells you that a Phytophthora is present in the sample.

A particular species of Pythium or Phytophthora will infect certain plants but not all plants. Each has what is called a 'host range', or list of plants that it infects.
A particular species of Phytophthora may be in your water but maybe you are not growing the crop it attacks.
A test kit would tell you that a Phytophthora is present but not give you enough information to evaluate whether that is important or not.
But some information is better than no information.

If the testing consistently is negative, perhaps the target pathogen is not present in the body of water being sampled.
If you get positive tests, then isolations have to be made and the species of Pythium or Phytophthora determined by a scientist.

A grab sample is taken at a specific time. If you use a bait, you leave it in the water over a period of time.
A number of things can be used as a bait.

►For example, rhododendron leaves can be used, especially for Phytophthora.

►It is best to use whole leaves and not cut or wound the bait. They can be put in a holder in the water.
If you cut or wound the bait, Pythium quickly invades the wound.
So if you are baiting for Pythium, it’s not a bad thing to wound the bait.

►After the deployment, the colonized part of the leaf can be cut out and plated on agar.

► In my lab, we use creeping bentgrass blades to bait for Pythium. But you could use leaf discs from rhododendron or from some other plants.

►We made something to hold the grass blades.
A holder can be simply a piece of plastic window screening, folded over and stapled closed with the bait inside.

►Apple and pear fruit, unwounded, are good baits for Phytophthora. Cucumbers are good baits for Pythium.
You can probably use the leaf or fruit from whatever crop is having the problem as a bait. How efficient in obtaining Pythium or Phytophthora will be a question.

►The bait is deployed for 48 hr. usually, retrieved, and then colonized tissue can be plated out for isolation or tested using one of the kits.

►In some cases, the bait is deployed for a week or more.

►Note that if the bait is not in a holder that excludes animals, turtles and other critters may eat the bait.

The advantage of a bait is that it is in the water exposed to the pathogen for a long time and exposed to a larger volume of water than what you can collect as a grab sample for testing.

►If you were concerned with a bacterial plant pathogen, you can use filtering to concentrate the bacteria but you would need a 0.22 µm pore size filter. You could then test that filter paper with one of the kits.
The kits for bacteria are for specific bacterial genus and a particular species within that genus.

If you are concerned with a virus, you would need to grow a plant that is susceptible to that virus in a manner that would guarantee that the plant is healthy. You could then irrigate that plant with water from the sample area you want to test and do that repeatedly over many days. You’ll have to wait for symptoms to appear. There are kits available for specific viruses.  Or, you can send the infected test plant to a lab to verify that it is infected with the target virus.

►Take away points:

Water is only one possible source of a plant pathogen. You need to have a good reason to suspect that water is the source.

Know which plant pathogen is causing crop losses
Decide which plant pathogen you are going to seek

You can't test for every plant pathogens in general. There are too many different ones.

Contact the person who will be doing the testing to work out details of sample collection, bait to use, and shipping requirements.

If you are planning on doing the testing, determine what test you can use.

Understand what each possible result means.
What does a negative test tell you and what exactly does a positive test tell you?

You probably should test the water repeatedly to increase your confidence that a negative test result truly means that the pathogen you are looking for is not present.

If what I have covered today sounds like a 'work in progress', you have heard correctly.
The science of detecting and identifying plant pathogens in water is progressing but there are big gaps in our techniques, especially those that can be used easily in the field.