Phytosphere Research

Zoospore collection system for nursery plants

Version 10/6/2020

Various methods can be used to detect root-infecting Phytophthora species in plants. We describe a method for testing leachate from container grown plants in our BMPs for Producing Clean Nursery Stock pages. The most practical method for monitoring large numbers of plants in a nursery involves capturing water that drains from the bottom of containers during irrigation and baiting that water with a Phytophthora-specific bait such as a green pear. This system allows for non-destructive testing of blocks of plants without requiring that the plants be moved from the benches upon which they are situated. Such testing can be done on a spot basis during an inspection, but more importantly, can be run by the nursery on an ongoing basis to monitor plants for infection. Drought tolerant plants growing in the nursery with frequent irrigation and low evaporative demand will commonly not show obvious top symptoms even when root rot is extensive.

Three pieces of equipment are needed to run this test:

This page discussed the construction of the first two pieces of equipment and how to set up the equipment to run a test.

Zoospore collection vessel (ZCV)

The design of this device is based on the behavior of Phytophthora zoospores and other inoculum leached from infected container plants. Zoospores swim upward, so they concentrate near the top of a water column. Root fragments and other debris that may contain sporangia or oospores either tend to float or settle to the bottom of a water column. To maximize zoospore concentration around the pear during the leaching period, a relatively tall and narrow container is needed. Phytophthora zoospores typically don't swim further than 10 cm in a static system, and chemical gradients that attract zoospores to the bait are likely limited to even shorter distances, so a small surface area is desirable. The vessel described below has an inner diameter of about 20 cm, so the edge of a pear floating near the center would be 10 cm or less from the vessel wall.

A large amount of water may be leached out of irrigated plants when running the leachate baiting test, so the vessel needs to concentrate inoculum from this large volume of water to optimize detection of Phytophthora inoculum by the bait. To accomplish this, excess water drains from the middle portion of the water column rather from the top or bottom where inoculum is most likely to accumulate. For this to occur, the vessel outflow needs to be sized large enough to accommodate the maximum amount of water inflow so all excess water will exit through the drain rather than flowing over the top rim of the vessel. A 1 inch (2.54 cm) inner diameter (ID) PVC pipe has sufficient flow to handle peak drainage from a set of plants covering about 10.8 sq ft (1.5 sq m) of bench. This is enough area to bait the maximum number of plants recommended in the test protocol.

The vessel we describe here may not be the most efficient possible collector. However, from tests we have conducted, it is clearly a fairly efficient design and has been used to detect Phytophthora inoculum in leachate water volumes ranging from less than 2 gal (7.6 L) (i.e., less than full vessel) to about 76 gal (288 L). It fits under nursery benches that meet the Phytophthora BMP standards and is made of readily procurable, relatively inexpensive, durable, and cleanable components that are easy to put together with simple tools. The insulated vessel wall is a plus, as it helps prevent excessive heating of the collected leachate in sunny conditions and maintains a more constant water temperature during the test. The flat spot at the base of the vessel (where the spigot is located) also simplifies installing the PVC fittings. If tests are conducted using this standard vessel, it is easier to make direct comparisons between results from different tests conducted following the leachate test protocol. Instructions for making a mini ZCV are given below.

Materials needed

Note: ID=inner diameter, FPT=female pipe thread, MPT=male pipe thread

Note - this design is a bit simpler and uses fewer pieces than our originally-posted design. It also provides a bit more free space at the bottom of the ZCV, which reduces the chance of the pear bait getting wedged between the pipe and the wall of the vessel.

Parts needed for zoospore collection vesselFigure 1. PVC fittings and pipe sections (all 1 inch diameter) needed for zoospore collection vessel. Existing spigot has been removed from the 2 gallon beverage container and a larger hole has been made. The pear exclusion device (cable tie) has been attached to the thread-slip elbow. Both a flat rubber gasket (off-white) and O-ring (black) are shown; only one of these is needed. Adjustable pliers and Teflon tape also shown.

Tools needed

What to do

See this video to see how to construct this vessel. The current the video has been updated to show construction of the newer ZCV design. The main steps are as follows:

Exterior view zoospore collection vesselsFigure 2. Exterior view of two assembled zoospore collection vessels. Labeling each vessel on the outside can be helpful for tracking when conducting tests.

Pear exclusion device for inside drain of ZCVFigure 3. After drilling two slots on opposite sides of the slip fitting end of slip-FPT elbow, a cable tie is threaded through to form a pear exclusion device that keeps the pear bait from obstructing the drain during the test. The end block and the free end of the cable tie are kept within the elbow to prevent them from creating wounds in the pear.

Top view of empty zoospore collection vesselFigure 4. Top view showing the inside of the assembled zoospore collection vessel. Note rubber O-ring between inside wall and elbow and pear exclusion device (black cable tie) over the elbow opening. Black lines visible on one of the internal ribs (10 cm above bottom) indicate the level to which water is drained before transferring it to a 1-gallon plastic zip-closure bag (see leachate test protocol).

Collection system (universal fit)

The collection system consists of the following components:

Materials and construction details for making a standard universal collection system are described below. The universal fit design is preferable if testing will occur in multiple nurseries or with benches of varying sizes because this collection system can be made to fit many different situations. It may not be the simplest or most efficient design for a specific bench. For repeated tests in a nursery with uniform benches, you may want to modify the collection system dimensions to achieve a better fit. It may also be more convenient to mount the collection system on a cart or frame that can be slipped under the benches.

Materials needed

Tools needed

What to do (making a single collection sheet)

corner holes for sheetsFigure 5. Holes in the corners of the collection sheets are centered 3.5 cm from each edge. A template made from a piece of flashing has been used to mark the hole locations (left). Holes can be punched with a 0.5 inch metal hole punch. At right, the punch has been chucked into a drill motor. When run at very low speed, the punch in the drill will cut the hole in the flashing. A piece of carboard is used beneath the vinyl to avoid damaging the punch.

vinyl flashing sheetsFigure 6. Collection sheet, showing grommets (inset and right) and placement of crease. Nylon cord with S hook can be tied through the grommet as shown for corners that will not need to be used to overlap with other sheets (the high corners of the upslope sheet and the lowest corners of the downslope sheet). Removable/linkable cords can be used on all corners as an alternative (Figure 9).

grommetsFigure 7. Installation of grommets into corner holes of collection sheets. The top portion of the grommet can be snapped in place with a 0.5 inch PVC tee with a short section of 0.5 inch PVC pipe in the tee joint. This method is a bit safer than using a hammer, which can damage the grommet if too much force is used.

making cord endsFigure 8. Steps in fabrication of cord ends from 3/4 inch wide PVC molding. The molding is ripped lengthwise down the center to produce PVC strips of the appropriate width, about 1 cm (top). At bottom left, holes are marked at 1.25 inch (3.18 cm) intervals on the center line of the PVC strips, before being drilled with a 0.161 inch diameter bit (4.1 mm or #19 drill). After the holes are drilled, the PVC strips are cut apart at the midpoint between the holes. Completed cord ends are seen in the background at lower left.

linkable cordsFigure 9. Configuration of linkable cords showing the S hook, cord lock, and cord end parts separately. The order of construction is: (1) loop the cord though the cord lock, (2) slip on the cord end and tie knots (3) pinch the S hook closed onto the loop in the cord (left).

inserting cord end in grommetFigure 10. The PVC cord end and knot in the end of the nylon cord will not slip though the grommet hole together, so the knot is pushed through first (top left and right) before slipping the PVC piece though the hole (bottom left). When linking two sheets together as shown here, the downslope sheet (right side, bottom right) will be under the upslope sheet.

Flow deflectors or water slidesFigure 11. The flow deflectors are simply strips of vinyl flashing with slits cut near the top to make two tabs. The tabs attach to the PVC pipe dams on the underside of collection sheet.

PVC dam V-notchFigure 12. Use a pair of metal shears or a saw to make a small V-notch at each end of the slit in the PVC dam. Dams are much easier to slip onto the collection sheets if this notch is present.

Setting it up

A video of the collection system in action during a test can be seen here.

single sheet across wide benchFigure 13. Installation of a single collection sheet spanning a 4 ft (1.2 m) wide bench from side to side.

two linked sheet across wide benchFigure 14. Installation of two linked collection sheets spanning 4 ft (1.2 m) wide benches from side to side.

single sheet along narrow benchFigure 15. Installation of single collection sheets lengthwise along narrow benches.

two sheets sideways along wide benchFigure 16. Installation of two linked collection sheets lengthwise along a wide bench. Sheets are sloped to drain toward the aisle.

PVC pipe dam for downslope endFigure 17. Slitted PVC pipe dams are easier to slide onto the edge of the flashing sheet if you start with the end of the pipe at the corner and slide straight down as shown in top image. Bottom image shows dams in place without a flow deflector attached. This image also shows three sheets used in series.

attachment to potsFigure 18. Cords can be hooked to containers if there are no places to hook on the bench top. The type of bench top shown here is not compliant with the Nursery Phytophthora BMPs because water can run along the boards, increasing potential for cross contamination. Note - this image shows our original hardware for linking multiple sheets.

block under ZCVFigure 19. Cinder block is used to elevate the zoospore collection vessel (ZCV). Sheet is mounted along the long axis of the bench. ZCVs are easier to access when along the aisle, but more care is needed to avoid knocking them over when pulling irrigation hoses. For this reason, it is helpful to point the ZCV outflow away from the aisle, as shown here.

Mini ZCV - A Zoospore Collection Vessel for small volumes of leachate

The mini ZCV can be used for collecting and baiting small amounts of leachate, from just over 2.7 L up to between 6 and 8 L. For volumes of 2.7 L or less, the drain used in this ZCV is not needed. For volumes more than about 6 to 8 L, use a standard ZCV (above). Tests that generate the amount of leachate appropriate for the mini ZCV include (1) bench leachate tests conducted with low numbers of small containers or (2) leachate type individual container tests on containers up to about #2 ("2 gal", about 6.3 L actual volume).

Materials needed

Note: ID=inner diameter, FPT=female pipe thread, MPT=male pipe thread

To assemble a mini ZCV, press the two elbows onto one of the PVC pipe sections so that they point in opposite directions (Figure 20). Hold the FPT (threaded) elbow opening against the middle of the outside of the plastic bag about 5-6 cm (2-2.5 inches) above the bottom seam of the bag. With your other hand holding the cut off riser inside the bag, line up the MPT threaded end of the riser with the FPT elbow and thread it in though the side of the plastic bag. The plastic bag will deform at the point that this is happening. When you finish tighening the riser, the slip-slip upper elbow should be near the top of the bag. As you tighten the riser into the elbow, the plastic may tear inside of the joint or it may simply stretch, leaving the connection plugged by a stretched layer of plastic. Insert a thin knife or similar sharp tool down the inside of the cut-off riser and cut away any of the plastic that blocks the threaded coupling. A flap of plastic can be left, but make sure that the hole is wide open so that flow will not be restricted.

composite photo of mini zcvFigure 20. Partially assembled mini ZCV. The cut-off sprinkler riser is threaded into the PVC FPT elbow through the side of the plastic bag. Cut away any of the plastic bag inside of riser-elbow joint that blocks flow though the joint, as described above.

Place the bag with the attached pipe into a second 1 gal plastic bag. This is needed to contain any minor leakage that may occur at the threaded joint. Then place the entire assembly into a plastic container that will support the bag when it is filled with water (Figure 21). Arrange the mini ZCV so that the cut off riser is at least 2 cm above the bottom of the bag. Insert the other length of PVC pipe into the upper elbow and extend it over the edge of the supporting container. Then pull up the top edge of the inner bag so that it extends at least 4-5 cm over the top of the upper PVC drain pipe. The outer plastic bag will need to be pushed or folded down so that drain pipe can be kept below the rim of the inner bag (Figure 21). At this point, the labeled pear bait can be placed into the inner bag and the mini ZCV is placed directly under the containers being leached or under a small collection system (Figure 21).

Figure 21. Left - Completed mini ZCV ready for use. Containers in this test drain directly into the inner bag. Center - Mini ZCV in use. A small, sloped vinyl sheet with creased edges is used to collect and direct water into the ZCV. Note overflow from the drain pipe (a second cut-off riser in this example). Right - mini ZCV after incubation with pear removed. The PVC outflow pipe was removed and placed between the 2 bags for transport. Note that the level of leachate in the bag is below the level of the upper elbow, preventing overflow though it.

After leachate collection is completed, tilt the mini ZCV slightly to allow the water level to drain about 2 to 3 cm below the outflow drain. Remove the upper PVC drain pipe; it can be stored in the supporting container or between the 2 bags for the time being (Figure 21, right). Adjust the mini ZCV so that the upper elbow is positioned well above the water line (Figure 21, right). Unfold the outer bag edge above the level of the elbow. Before moving the mini ZCV, close both bags to prevent splashing. When the mini ZCV has been moved to where incubation will occur, open both bags, making sure that the top edges of both bags are above the top PVC elbow. After incubation is completed and the pear bait has been removed, the cut-off riser can be unscrewed from the pipe and these parts can be sanitized with freshly diluted bleach (0.5% sodium hypochlorite for at least 2 minutes) for later reuse.

10/06/2020 - Added new link for updated ZCV construction video and made a few assocaited edits where ZCV assembly is discussed

12/11/2019 - Added link for directions for constructing an irrigation wand to equipment needed list.

9/29/2019- instructions for making the mini ZCV added

4/30/2019- expanded the writeup with additional pictures and details, modified design of ZCV and collection sheet attachments to make construction simpler.

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