Plants in a nursery well spaced and raised on metal benches

Plant Testing for Phytophthora

Plant Testing with the AIR Program

Plant testing is an integral component of accreditation with the AIR Program. Testing provides a crucial check on BMP compliance in the nursery, and is required both to achieve and maintain accreditation.

The standard testing method used by the AIR Program is the Bench Leachate Test using pear baiting to detect Phytophthora zoospores present in irrigation flow through from batches of container plants produced in the nursery. During initial accreditation site visits, AIR team members will demonstrate how to conduct this test with nursery staff. Once accredited, nurseries are expected to perform their own periodic testing using this method and upload records prior to reaccreditation.

Two sets of leachate testing equipment. Container plants on nursery benches over collection sheeting and jugs of water.
Figure 1. In-progress leachate tests, setup with a complete leachate collection system, single block of test plants, and a floating pear bait. 

Jump To

Bench Leachate Testing

In essence, leachate testing for Phytophthora consists of irrigating container plants with a defined volume of water over the course of six irrigations, then baiting the collected leachate water for the presence of Phytophthora zoospores. These flagellated zoospores, capable of detecting plant matter and swimming though water to new hosts, are attracted to pear baits placed in leachate water. Once infected, these pear baits develop recognizable lesions indicating presence of the pathogen in tested arrays of plants. 

Container plants in racks on a bench, with water being applied to each container individually
Figure 2: Irrigation using a calibrated watering wand. Plants are placed directly about the Runoff Collection Sheeting such that water drains into the Zoospore Collection Vessel. Photo credit: Phytosphere Research

Instructions

  1. Sanitize benches with 10% bleach (0.525% Sodium Hypochlorite, 5000ppm available chlorine). Allow contact for at least 1 minute to ensure adequate sanitation time, then rise well (Table 1).
  2. Set up Zoospore Collection Vessels (adapted water cooler) and Runoff Collection Sheeting. Collection Vessels should be positioned such that flowthrough leachate water directly fills the vessel without splash or significant loss of water.
  3. Select plants for leachate testing. We recommend biased testing based on visual symptoms or relative risk. Note batch information, including the species tested, number of containers included, number of containers in the batch, and any test notes such as suspected symptoms or batch number.
  4. Based on your selected plants, find your required irrigation volume for each batch based on pot size.
    • Irrigation volumes and flow rates for common pot sizes can be found on the provided tables (see Tables 2 and 3).  
    • If your required pot size by volume is not included, you may approximate your appropriate irrigation volume by multiplying the volume of your container (in milliliters) by 17.5%.
  5. Arrange plants on the bench directly above the Runoff Collection Sheeting, such that all leachate water flows down the sheeting into the collection vessel (Figures 1 and 2).  
    • Note: A maximum of 40 plants per batch may be included in a single test. For larger blocks, select a representative batch of 40 plants per block using biased sampling of symptomatic or poor plants and plants along edges of the block.  
  6. Using clean disposable gloves, place your fresh pear and thermometer in the leachate collection vessel.
  7. Calibrate your watering wand to the appropriate flow rate. By calibrating to a specific flow rate (in milliliters per metronome beat), you can apply a standardized volume of water to each container by counting beats during irrigation.
Right:A zoospore collection vessel filled with water. Left: water draining from the vessel through the pour spout.
Figure 3. Proper drainage from the bottom of the Zoospore Collection Vessel. Photo credit: Phytosphere Research
  1. Note the start time and begin irrigation, following the metronome to ensure each pot is supplied with the same amount of water. If running multiple tests, continue down the line until each batch has been irrigated.
  2. After 15 minutes, repeat the irrigation. Continue in this fashion until each batch has received 6 irrigations. Record each irrigation time.  
    • Note: The entire applied volume must flow through the pot. As soil becomes saturated pots with little headspace may begin to overflow. If this occurs, split each irrigation to prevent overflow. For example, if you apply 110ml in 4 beats, you may split this irrigation into 2+2 beats ( applying 55 ml in each of the 2 beats) , such that each plant is watered twice per irrigation to apply the total final volume.
  3. Allow 15 minutes after the final irrigation, then pull the pear from the leachate water and place in a labeled Ziplock bag. Record the time and the final water temperature.  
A man holding bag with a pear inside next to a leachate collection vessel filled with leachate water.
Figure 4. Collected leachate ready to be poured into a Ziplock bag and bucket for transportation and incubation.
  1. Drain excess leachate water by tilting the overflow spout until the water level reaches the marked line.   
  • Note: Zoospores collect at the top of the vessel. Emptying the water from the bottom of the vessel thus allows us to concentrate spores in the remaining water.  
  1. Pour the remaining leachate water into the Ziplock bag (Figure 4).
    • Note: A single round of testing will take an hour and a half, not including setup and cleanup time.

Single Plant Root Baiting

In some cases, single plant testing of symptomatic plants may be appropriate rather than testing full batches of plants using the Leachate Testing Method.  If plants exhibit Phytophthora root rot symptoms including wilting, stunting, leaf necrosis, root discoloration, root rot and reduction of root biomass, they will be good candidates to conduct this testing.  Root baiting of individual plants provides a quick and easy test for the presence of Phytophthora and other oomycete species in symptomatic plants and culls. While less robust than leachate testing, it can be used as a quick check for symptomatic plants. 

Note: root baiting should not be used as a replacement for standardized, full-scale leachate testing but rather as an additional tool in your Phytophthora-exclusion toolbox. It is also a destructive test, so plants tested via this method are typically no longer viable after testing. 

Instructions

a pear floating in a bucket of water with a plant
Figure 5. Root Baiting for Phytophthora
  1. Select symptomatic plants for testing.
  2. Prepare a bucket by cleaning with soap and water and/or spraying with 70% isopropyl alcohol. Allow to dry before use. Alternatively, you may line the bucket with a 1-2 gallon Ziploc freezer bag.
  3. Remove the symptomatic plant from its pot. Examine the roots and note any discoloration or dieback. Discolored or necrotic roots may be a symptom of Phytophthora infection or infection by another root rot pathogen.
  4. Place the symptomatic plant with roots and soil in a clean bucket and fill with water to just above the soil line. The bucket should be large enough to easily accommodate the full plant and soil with some space, but not so large that there is excess water.
  5. Place a clean, unbruised pear in the bucket. Check to make sure that the pear floats at the water line for the best chance of zoospore capture. If the pear sinks, you may attach a piece of foam or other buoyant material to the pear using a rubber band (Figure 1).
    • Occasionally, soil mixes high in perlite and/or coir may have excessive debris floating at the top of the bucket that may burry the pear. This excess debris may be removed so that the pear is able to float in the bucket. 

Pear Bait Incubation

Once the leachate test has been completed, the pear bait must incubate typically for 2-5 days before lesions develop. Lesions may develop while still incubating in leachate after only 2-3 days, or up to 8 days. Pear bait lesions typically present as dark, chocolate brown lesions which may or may not be hard to the touch. 

It is important to note that not all lesions that develop are due to Phytophthora. Occasionally, lesions may develop due to other oomycete species such as Pythium, often but not always associated with breaks in the skin, or contaminating microbes. For this reason, it is important to send pears showing lesions to a lab for diagnosis.

a pear showing dark brown lesions caused by Phytophthora
Figure 6. A pear showing lesions due to Phytophthora infection.

Instructions

  1. Allow the pear to incubate in the leachate water for up to 3 days. Leave the bag open to allow aerobic conditions. Lesions may form at this stage in some cases.
  2. After 3 days, remove the pear and allow to incubate at room temperature for up to 5 days. Lesions may develop as soon as half an hour after removal from the water, or they may take several days.
    • Note: If left too long, secondary infections or Pythium may overtake the pear and mask positive results.
  3. Pears with distinctive lesions should be considered suspect for Phytophthora (Figure 6). To confirm Phytophthora detection, pears should be sent to a laboratory for diagnostic testing.

Note: A negative test means that Phytophthora was not detected by the current round of testing. However, keep in mind that negative detection does not necessarily mean that plants are Phytophthora-free! If plants are destined for use in highly sensitive restoration projects, for example, a second round of testing should be considered before out planting.

Resources

For printable instructions on how to conduct the Bench Leachate Test, as well as how to construct testing equipment and perform single container root baiting tests on symptomatic plants, please review the following handouts:

a petri dish with Phytophthora colonies growing from plated pear skin samples
Figure 7. Pear lesion tissue assay showing growth of Phytophthora.

 

Pear baits that develop lesions should be sent to a lab for diagnosis to confirm pathogen identity. The Del Castillo Lab at UC Davis provides this diagnostic service to AIR-participating nurseries free of charge. For instructions on how to submit symptomatic pear baits for identification, please see the Del Castillo Lab Website, linked here.

Documents