Testing Procedures
for BMPs for Producing Clean Nursery Stock

3.4.  Bench testing leachate from batches of container nursery plants

Version 10/9/2018

Application:  Testing blocks of plants on a bench (or cart) to determine if any plants are infected.  Tests the entire root systems of all plants in the test area for the presence of Phytophthora by detecting zoospores released from sporangia on the root system during repeated irrigations.

Advantages:  Allows testing of many plants at once.  Can be conducted without moving plants from benches.  Plants do not have to be unpotted.  Nondestructive and does not expose other plants to inoculum beyond what may occur during irrigation.  No false positives if bait spots are confirmed by culturing.  Culturing can identify Phytophthora species present. 

Limitations:  Positive reactions require at least several days to develop and can take longer to confirm depending on methods used (Figure 3).  Baiting conditions (temperatures, irrigation history) can affect test sensitivity.  Previous application of fungicides (systemic oomycete suppressive chemicals) to tested plants may interfere with test. No single bait is optimal for all Phytophthora species.  Does not identify which plants or how many plants within the tested block are infected.  This protocol is designed for plants that have been regularly irrigated up to the time of testing.  It may not be effective for plants that have been dry for an extended period because sporangia may not be present in such material.

The standard (short duration) protocol described below has detected a variety of Phytophthora species in container-grown nursery plants across a wide range of temperatures and in a variety of test situations. In tests conducted to date, Phytophthora has been detected in batches with known infection rates as low as about 2.5% to 5%, but detection efficiency is influenced by the infection level and root density of the Phytophthora source plant. Further tests are being conducted to determine how the detection threshold (minimum number of infected plants needed to obtain a detection) may be influenced by container types, Phytophthora species, plant species, and temperature ranges present in nurseries.

Recommended practices: Based on data collected to date, the following practices are recommended to maximize the sensitivity of this test:

Equipment:  This method is based on collecting irrigation water that drains out of a set of nursery plant containers and directing it into a specialized collection vessel that contains a bait.  The two main components of this system are described below. 

Runoff collection system:  Use a smooth, easily sanitized, inert material that will fit under the benches that will be tested.  Vinyl flashing works well (available in rolls of varying sizes – we have used the 0.5 m [20 inch] width); the glossy side should be used for the collection surface. Multiple sheets can be overlapped to obtain sizes needed for varying bench lengths and widths. The collection surface can be suspended beneath a bench using nylon cords. The cords tie or hook to the sheet on one end and are hooked to the bench or pots on the bench on the other end (Figure 4). We use plastic cord locks to help adjust the lengths of the cords. The collection surface needs to be sloped to drain to one end and the flow needs to be channelized so that all of the water will run into the collection vessel (Figure 4). See this link for instructions on building the collection system in Figure 4. The outflow from the runoff collection sheet should be set as close as possible to the top of the collection vessel to minimize splash and turbulence in the collection vessel. You can use a strip of vinyl flashing (8-10 cm wide) to bridge the gap between the collection sheet and the vessel below it, which will allow water to flow with less splash and turbulence.

Zoospore collection vessel (ZCV):  This vessel is designed to optimize detection of zoospores.  Zoospores swim upward and should concentrate near the top of a water column. Debris that may contain sporangia or oospores tends to either float or settle to the bottom of a water column. The collection vessel is designed so that excess water drains from the lower middle portion of the water column. Water in the vessel enters an open pipe elbow at a height of about 7 cm (2.75 inches) above the inside bottom of the vessel. The water flows out through an elevated external pipe that will leave the water level in the vessel about 6 cm (2.5 inches) below the rim (Figure 5). The diameter of this drain pipe should be large enough to prevent water from overflowing the top of the vessel during peak water flow from the irrigated pots. An internal diameter of at least 2.5 cm (1 inch) is sufficient to handle peak flows from a set of plants covering about 1.5 m2 (10.8 ft2) of bench.  The vessel should be tall and relatively narrow to maximize zoospore concentration near the top.  A modified 7.6 L (2 gallon) insulated plastic beverage jug with an internal depth of 30 cm (12 inches) is illustrated in Figures 4, 5. See this link for instructions on building the collection vessel in Figures 4 and 5.

For testing small batches of small containers, which generate low amounts of leachate (generally less than 8 L L or 2 gal) and low runoff rates, we have sucessfully used a "mini ZCV", shown in Figure 8. See this link for instructions on how to construct the mini ZCV. Other possible vessel designs (such as wider or more elongated vessels) have not been tested and may not be as efficient for baiting, so we recommend using a vessels as close as possible in size and shape to these two designs.

Methods:  Standard baiting uses green, nonwounded pears, which can be infected by a wide variety of Phytophthora species. Most pears will float in water. If the pear sinks, use a different pear or make a pear floatation device (PFD) for it by using a clean rubber band to attach a small piece of closed cell foam (e.g., a packing peanut) to the pear. Leaf baits can also be used if you have arrangements for testing these baits.  You will want to make arrangement to keep these floating on the water surface. See 3.2.  Detection by baiting – general procedures for more information on choosing pears and baiting procedure details.

Pre-test conditions: Plants to be tested should have been receiving regular irrigation prior to testing. Sporangia may not be present in the soil of plants that have been dry for an extended period. Testing should be done when average soil temperatures have been in the range of 18-24°C (65-75°F) for at least three days, preferably a week or more. These temperatures are favorable for sporangium production in a wide range of Phytophthoraspecies. It is possible to detect some Phytophthora species at temperatures outside of this temperature range, but detection efficiency for many species may be reduced when soil temperatures are well above or below this range.

Water temperature: The temperature of irrigation water applied during the test should no higher than about 22-25 C (72-77 F) if possible. We have successfully baited Phytophthora niederhauserii (growth optimum 30 C [86 F] maximum 37 C [99 F]) using warmer irrigation water, but not all Phytophthora species will be successfully baited at very high or very low temperatures. If temperatures are high and hoses have been lying in the sun, run the water until it cools to a stable acceptable temperature before applying it to the plants.

Standard (short duration) protocol.  Suspend the runoff collection system beneath a mesh bench or cart that has been washed to remove debris, sanitized by rinsing with a fresh 0.5% NaOCl (diluted bleach) solution, and rinsed with clean water to remove all bleach residues. If you will be reusing the same space to test another batch of plants, this sanitation process needs to be repeated before the next test, along with cleaning and sanitizing the runoff collection system and zoospore collection vessel.

Place a labeled, unwounded, green pear (with a PFD if needed) in the bottom of the zoospore collection vessel before starting irrigations.  Water should be applied individually to every plant container in the test batch, not simply sprayed over the top. Irrigation is applied to avoid overflow from container rim. This is most easily accomplished using an irrigation wand with an on/off lever or trigger. It is also helpful to have a flow control at the wand. Otherwise, you will need to control the water flow at the hose bib. Finally, for small containers in trays or racks (such as Deepots), it is useful to have an irrigation wand that can produce a narrow stream of water that can be directed into each container. The objectives the the test irrigations are to:

Any water that does not flow through the container media will only dilute the spores in the collection vessel unnecessarily and can reduce the sensitivity of the test. Applying equal amounts of water to all plants in the test minimizes bias associated with drainage rates or the amount of headspace in individual containers, which are not necessarily related to their risk of being infected. To the degree possible, avoid excessive splashing between containers in the test; some splash between containers in a close-packed test array is unavoidable.

Under hot or sunny conditions, be sure to check the temperature of the water coming out of the wand before each irrigation event. If hoses or aboveground pipes are exposed to sunlight, the initial water temperature may be very warm, so run it until it cools before each irrigation event (see Water temperature above).

Apply water in six irrigation events at 15-minute intervals. At each of the six irrigation events (including the first), enough water should be applied so that water will drain from the bottom of each container. If the potting mix in the containers is somewhat dry before the test is started, it may be necessary to apply an additional dose of water at the first irrigation event so that drainage from the containers occurs by the end of that event.

Irrigation volume:The amount of water that can be applied to a container before overflowing the container rim is limited by the amount of headspace (distance between the soil line and the pot rim), the drainage rate of the potting media, and the flow rate from the irrigation wand. It is easier to apply the target amount of water per irrigation if containers have sufficient headspace (at least 1 cm for small containers, several cm for larger containers). If there is very little headspace in the containers, you will need to use a very low flow rate and/or apply water to each container in two (or more) doses for each irrigation event. Apply the first dose of water to each of the containers in the test and then apply the second dose after the water has drained out of the headspace.

Based on tests we have conducted, recommended application volumes for each of the six irrigation events are about 15-20% of the nominal container volume per irrigation. The table below shows approximate water volumes to apply to containers of different sizes. If using another type of container, calculate the amount needed per irigation event by multiplying the actual container volume by 17.5%. To deliver these fixed amounts to each container, adjust the irrigation wand to a suitable flow rate and determine how long it takes to dispense the required water amount into a calibrated container (e.g., a graduated cylincer or measuring cup). You can measure the time by counting at a constant rate (e.g., a count of 5) or use a pacer or metronome application to provide an audio pulse (e.g., 4 beats at 60 bpm = 4 seconds). If containers are spaced tightly, you can use a continuous flow as you move from container to container based on your count or audio beat. If containers are more widely spaced, or you need to move between spaced racks, stop the water flow between containers.

Container type Container volume, ml Irrigation volume, ml Percentage
Ray Leach "Cone-tainer" SC7 Stubby 107 20 18.7%
Treeband 2 x 5 inch 393 70 17.8%
Deepot 40 655 100 15.3%
Treeband 4 x 10 inch 2622 500 19.1%
Treepot 4 x 14 inch 2650 500 18.9%
#1 ("1 gal") 2839 550 19.4%
#5 ("5 gal") 14385 2500 17.4%

Especially for larger plant batches or containers that drain rapidly, check to see that the water flow rate does not exceed the collection vessel’s outflow rate. If the water in the vessel gets too close to the rim, pause irrigation periodically.

Mark the start of irrigation and record each start time on your data sheet (an example data sheet is here). Starting a 15 minute timer at the start of each irrigation can help you keep track of when to irrigate. The collection period will take at least 1.5 hr from the start of the first irrigation. Check to make sure that the pear is floating once the water level is high enough. It may get wedged between the ZCV wall and the internal drain pipe or under the water deflector. If necessary, keep the ZCV shaded to prevent excessive heating of the water in the vessel.

After the last irrigation, let the containers drain for at least 15 minutes. Allow a longer time if substantial water drainage is still occurring; the flow from the containers should be stopped or nearly stopped. Wearing clean gloves, remove the pear and place it into a labeled 1 gallon heavy-duty plastic bag (e.g., 1 gallon Ziploc® freezer bag) that is supported in a plastic container. Slowly drain excess water from the center of the water column through the outflow by pivoting the outflow downward (Figure 6) or tilting the collection vessel. Then pour the remaining water, which will include both surface water and soil/debris from the bottom of the vessel, into the plastic bag. A one gallon freezer bag will hold about 2.7 L. The bag can be mostly full, but leave at least 7-8 cm (3 inches) of space near the top so the bag can be sealed for transport and to prevent spillage. Leave an air bubble in the top of the bag when you seal it for transport. Leave the bag open in a shaded area (preferably no warmer than 27 C [80 F]) where it is not subject to cross-contamination if it will be held for an extended period (more than 1-2 h) before transport to the incubation chamber/room (Figure 7). If placing in a cooler for transport, avoid subjecting the samples to a temperature shock, which may cause zoospores to encyst.

Incubate and evaluate pears as discussed in 3.2.  Detection by baiting – general procedures.  The plastic bag should be opened to allow air exchange during incubation (Figure 7).

Phytophthora lesions typically appear on baits no sooner than about 2 days after the end of the leaching procedure. Most lesion will show up within 5 days after the leaching procedure, but in some cases, symptoms not become obvious until 8 days. If no Phytophthora symptoms are apparent 8 days after the leaching procedure, test results are negative (i.e., no detection). The timeline for testing is summarized in Figure 3.

Note: With multiple sets of equipment (collection systems and ZCVs) and adequate bench space, multiple tests can be run in parallel. Allow adequate separation to prevent any cross-contamination via splash between different test sets. Typically, a single person can irrigate up to 6 sets of test plants within the 15 minute irrigation intervals. Irrigation time for each set of plants can be affected by the number of plants in each test array, plant density (more difficult to direct water past foliage to container in dense or spreading plants), headspace (more time needed to apply multiple doses if headspace is low), container size, or other logistics (having to move hoses, etc.).

Post-test equipment sanitation.  Rinse all equipment thoroughly and sanitize with diluted bleach (see Phytosanitary Procedures for BMPs for Producing Clean Nursery Stock) followed by a thorough rinse with clean water to remove residual bleach before reusing equipment for tests on another set of plants.

Long duration protocol.  The long duration baiting protocol has been tested only under a limited set of conditions, so its sensitivity under a variety of conditions is unknown. Set up the collection system and place a labeled unwounded green pear in the bottom of the vessel. Irrigate plants thoroughly until the collection vessel is filled. Irrigate plants at least once daily as described above, preferably twice a day. Leave the pear in place until symptoms develop (a minimum of 2.5 to 3 days) or remove after 5 days if no symptoms are seen. 

Incubate and evaluate pears as discussed in 3.2.  Detection by baiting – general procedures.

Results:  Confirmed positive results (Phytophthora lesions confirmed by culturing, sporulation on water-floated lesions, or PCR) indicate that at least one of the tested plants in the block is infected. In a closely-packed block of plants, it is likely that multiple plants are infected. Adjacent blocks of plants should be considered to be at risk of infection, but may not provide positive results if the plants have been infected only recently.

Negative results from a single test should be interpreted with caution. At least one more test conducted at least one week after the first negative test should be conducted to provide a higher level of confidence, especially for plants to be used in sensitive habitats. If initial negative tests were conducted under very cool or warm conditions, additional tests should occur under more moderate temperatures. Greater confidence can be associated with negative tests from nurseries that are stringently following the procedures outlined in Best Management Practices (BMPs) for Producing Clean Nursery Stock.

timeline for bench test

 Figure 3.  Timeline showing sequence of activities for conducing bench test of leachate for Phytophthora species using green pear baits.

Armstrong 3 sheet test setup

 Figure 4. Bench test setup variations. A: Three overlapping sheets of vinyl flashing are held in place beneath the bench using nylon cord with S hooks. Cord tension is adjusted with cord locks to obtain a sloping surface across all sheets that will drain into the collection vessel.

Armstrong 3 sheet test under bench view

Figure 4B. When plants are irrigated, water draining from the pots is directed into collection vessel, which contains an unwounded green pear as bait.  Slit PVC pipe sections at edge of flashing near the collection vessel provide rigidity and help direct flow.

2 sheet test setup

Figure 4C. Collection systems set up with 2 vinyl flashing sheets in parallel on a bench. Sheets are sloped to drain toward the aisle.

1 sheet test setup on narrow bench

Figure 4D. Collection system set up with 1 vinyl flashing sheet on a narrow bench.

1 sheet test setup on cart

Figure 4E. Collection system set up with 1 vinyl flashing sheet on a cart. Cart is wide enough for two such test arrays. Arrays drain in opposite directions to minimize potential for cross-contamination.

zoopspore collection vessel with pear exclusion device

Armstrong bench bait test_20160217_07.JPG

Figure 5.  Collection vessel for bench test.  Top image shows the height differential between the internal drain pipe and the external overflow, which maintains the water column below the rim as water enters the vessel (bottom).  A guard over the internal drain (top) prevents pear from plugging the drain, which may occur if the pear is forced down by incoming water.  A pool thermometer (top) is used to monitor water temperature.

tilting outflow to lower water level

Figure 6. Tilting the outflow of the zoospore collection system will allow water to drain from the center of the water column. The objective is to conserve the water from the top of the water column as well as the water and debris at the bottom; this conserved water will be transferred into the bag with the pear bait. Mark the inside of the vessel at the 2.7 L level (small black line below water level) to simplify draining the container to the level that will fit in the bag (heavy-duty 1-gallon zip closure plastic bag).

pear baits in bags for incubation

Figure 7. After the 1.5 hr leaching period, transfer the pear bait and about 2.7 L of water from the ZCV into a 1-gallon zip closure plastic bag. A heavy-duty (freezer) bag is preferable; double bag (left) if using regular bags. Each water-filled bag is placed in a rigid-sided container (cut-off bleach bottle or similar) that is as tall as the water level in the bag. After filling bags, leave them in a shaded location with bags open (left) until ready for transport. Seal bags whenever they are moved to prevent spillage. After transport, plastic bags are left open for the 3-day incubation period during which the pears float in the leachate (right - see also instructions for building a simple incubator).

mini zoospore collection vessel

Figure 8. A mini ZCV can be used for test arrays consisting of few and/or very small containers (center, right). Such an array does not generate very much leachate during the test so a smaller ZCV can be used. The mini ZCV uses the same design principles used in the standard ZCV but a heavy-duty (freezer) 1-gallon zip closure plastic bag is used in place of the plastic jug (left).

Updated 10/9/2018 - more details on standardizing irrigation volumes by container volume, with assocaited edits and some reorganzation. Add description and image of mini ZCV.

Updated 8/22/2018 - additional details added to standard protocol irrigation procedures, including guidance on irrigation volumes.

Updated 8/19/2017 - expanded explanations, added more photos, a figure illustrating timeline of bench baiting process, and a link to sample datasheet for bench testing.