Testing Procedures
for BMPs for Producing Clean Nursery Stock

3.3.  Individual plant sampling/baiting

Version 2/28/2019

Application:  Testing symptomatic or suspect individual plants.  Tests entire root system of individual plants for the presence of Phytophthora by detecting zoospores released after a flooding period.

Advantages:  Fairly sensitive test of entire root system, relatively easy to conduct, plant does not have to be unpotted.  Nondestructive and does not expose other plants to inoculum.  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.  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.  Not suited to testing large numbers of plants.  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 dead or dry for an extended period (e.g., nonirrigated culls) because sporangia may not be present in such material.

Methods: Standard baiting uses green, nonwounded pears, which can be infected by a wide variety of Phytophthora species.  Leaf baits can also be used if you have arrangements for testing these baits. To minimize the possibility of cross-contamination between plants, wear waterproof gloves when handling containers and pear baits. Sanitize gloves before handling a different plant or other items assocaited with them. Read 3.2. Detection by baiting – general procedures and Using green pears to bait for Phytophthora to learn important methodological recommendations.

There are 3 methods that can be used for individual plant baiting:

  1. Direct baiting of the entire plant
  2. Leaching of the plant while in the container
  3. Flooding of the plant in the container

1. Direct baiting of the entire plant: This method can be used for individual plants that are recently dead, or in severe decline, if the plant and soil of the container are not too large to fit readily into a gallon ziplock bag. Cut off the top of the plant. The root system and soil should not take up more than about 1/3 of the space in the bag. Place the bag into a rigid sided container to provide support and place a labeled green pear onto the soil surface. Add water to the bag - charcoal filtered tap water is preferred if available, until the soil surface and root system are covered by water about 1 inch deep. Incubate and evaluate pears as discussed above in 3.2. Detection by baiting – general procedures and Using green pears to bait for Phytophthora.

Figure 2. Direct baiting of an entire plant in a one gallon zip close bag. Pear has been shifted from its original position to show Phytophthora lesions that have formed on the pear.

2.Leaching of the plant while in the container: This method can be used for testing plants in a range of container sizes. It is basically a single container version of the bench testing leachate test protocol. Read and follow the directions given at bench testing leachate test protocol to conduct the test. Place the container to be tested over the runoff collection system. This can be a sheet of vinyl suspended below the bench under the plant, a large plastic bag, or a small plastic bag, depending on the volume of water that will be generated by the test. Use Table 1 at bench testing leachate test protocol to estimate the amount of water that will be generated by the test, which will approximately equal the container volume shown in column 2. To collect runoff from the test, use either a standard zoospore collector vessel (ZCV), a mini ZCV, or a plastic bag supported in a small container if all leachate generated by the test and a green pear will fit into the bag. Minimize the distance between the container and the collection vessel to minimize splash and reduce turbulence.

Figure 2a. Leachate method being used on individual 5 gallon container plants. In this photo, leachate is being collected in mini ZCVs. The plastic bag functions as the runoff collection system. A hole in one corner of the bag is placed so that runoff drips through the hole into the ZCV. Floating pears can be seen in some of the mini ZCVs.

Figure 2b. Leachate method being used on individual D40 plant. Bungee cord holds pot in place on metal frame, leachate is being collected in a 1 gallon zip-close bag. Green pear visible in bag.

Figure 2c. Leachate method being used on individual 1 gallon plants. Plastic mesh holds pots above 2.5 gallon containers baited with green pears. The approximately 1 gallon of leachate and pear for each test plant will be transferred to plastic bags supported in cut off plastic bottles after the test irrigations are completed.

3. Flooding of the plant in the container: This method works best for 1 gallon or smaller stock, larger container sizes are awkward to bait in this manner. Place the container of each plant to be tested in a heavy-duty plastic bag (e.g., 1 gallon Ziploc® freezer bags for 1 gallon pots or smaller stock).  If the container has enough room on top, use a clean waterproof glove to make a slight depression in the potting media in the top of the pot and place a rinsed, labeled, unwounded green pear in the depression (Figure 2d). Avoid scratching the pear if the potting media contains abrasive components such as perlite or lava rock by setting it lightly in the depression. If using a smaller container, place the pear in the bag alongside the container (Figure 2e). Leaf baits, if used, are added to the top of the container or the bag at this time. Irrigate the plant with clean water until water accumulates in the bag up to the depth of the top of the soil (Figure 2d,e).   Water temperature should be in the range of 18-24°C (65-75°F).  Do not expose the flooded plants to direct sun that could allow the water to heat up excessively. 

Leave plants flooded in the bags for one to two hours.  At the end of this period, remove the pot from the bag and allow the excess water to drain from the pot into the bag.  You will need to support the bag by placing it in a plastic container, such as a cut off 1 gallon plastic bottle (Figure 2f).  Collect drain water from the plant container until dripping has stopped or nearly stopped.  The total water volume from each pot will vary by pot size, but typically should be between 1 and 2.5 L (about 1-2.5 quarts).  If the pear bait was in the top of the container, transfer it to the water in the bag using a clean waterproof glove immediately after the pot is removed.  Wash, sanitize, and rinse your gloves before handling another test plant. 

This test can also be conducted by flooding the plants as described above and adding the pear bait at the end of the flooding period. However, in plants producing low amounts of inoculum (e.g., recently-infected plants), adding the pear at the end of the flooding period may reduce test sensitivity.

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

Results:  Confirmed positive results provide proof that the tested plant is infected.  Adjacent plants should be considered to be at risk of infection.  They may not provide positive results if the plants have been infected only recently.  Negative results from a single test should be interpreted with caution. 

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.  If plants are targeted for use in sensitive or other non-infested sites, a second test conducted at least 1 week after the first, and testing with additional bait types, should be conducted to provide a higher level of confidence.

individual pot baiting 1 gal

Figure 2d. Baiting of individual flooded plants.  Water is added to pot until water level is at or slightly above the top of soil in the pot. Pears have been placed in each container before flooding.

individual pot baiting with pear in bag

Figure 2e. Baiting of individual plant by flooding. For small containers, the pear bait is added to the bag rather than placed into the container top.

pear bait in leachate Phytophthora infected pear bait

Figure 2f.The bait is incubated in the water (left) until symptoms develop or for up to 3 days.  Pear bait on right was removed 2 days after being placed in water from an individual pot test of a Ceanothus and shows extensive early symptom development.  Note that most of the of the infections are associated with lenticels in this pear.

Updated 8/24/2017 - added images, updated to show adding pears to pots/bags during flooding period.

Updated 1/27/2019 - added methods for whole plant baiting and individual container leaching and associated images.

Updated 2/28/2019 - added additional images.