Testing Procedures
for BMPs for Producing Clean Nursery Stock

3.2.  Detection by baiting – general procedures

Because Phytophthora species can be difficult to isolate from diseased plants, plant pathologists often use baits to detect Phytophthora.  These include seedlings, leaves, or fruits of various host plants (Erwin and Ribeiro 2005). Different baits vary in their susceptibility to the various Phytophthora species, and no single bait can detect all species. Among baits, green pears are readily available, are susceptible to many common and uncommon Phytophthora species, and are relatively easy to interpret. Pears can be used to detect Phytophthora in soil samples, water samples, and root samples.  A negative result using pear baits may result if the Phytophthora species present does not readily infect pears.

Baits need to be added to samples when actively swimming zoospores are likely to be present (see Plant nursery conditions favor Phytophthora root rots).  If zoospores encyst before baits are added, baiting may provide a false negative result.  Depending on temperature, a given zoospore will generally swim no more than 8 to 24 hours.  Mechanical agitation and rapid changes in temperature or the salt concentration of the water can induce zoospores to encyst.

3.2.1.  Selecting and preparing pears for baiting

It is important that pears be as free of wounds and as green as possible.  Smooth, green-skinned varieties (e.g., Bartlett, D' Anjou, Packham) are all acceptable, but green D’Anjou pears tend to be slower to ripen, which is desirable.  Phytophthora species are among the few organisms that can infect green, unwounded pears. Ripe pears and those with nicks and scratches are susceptible to invasion by organisms other than Phytophthora, especially Pythium species.  Don’t use pears with large wounds and avoid pears with many wounds, especially fresh wounds.  If there are small surface wounds or discoloration, you can use a permanent marker to make a light dotted line around the affected areas, which can help you interpret what you see when pears are removed after baiting.

Rinse pears thoroughly before using. You can also wash pears using a little dish soap, but be sure to rinse thoroughly. Using a permanent marker, number the pear with the sample number.  Don't try to peel off the grocery sticker, as it will generally tear the pear skin.

3.2.2.  Incubating pear baits

Pear baits in water samples should float at the water surface.  If pears do not float, flatten the bag or transfer the water and pear to a clean, wide container in which the top of the pear will be at or slightly above the water surface.  Alternatively, use a different pear or make a pear floatation device (PFD) for it from a small piece of closed cell foam (e.g. a packing peanut) and a clean rubber band. Bags should remain open to allow for air exchange, but close bags while transporting to avoid spills.  Make sure that the bag is in a container (e.g. cut-off 1 gal plastic bottle) that will not allow the sides to collapse and spill water.  The container should be large enough to prevent any accidental spills or leakage if the bag is punctured.  Place these containers a secondary container (e.g., a plastic tub) if necessary to contain spills.

Incubate pears in water samples at moderate room temperatures.  A fluctuating day/night temperature regime ranging between about 18-24°C (65-75°F) is generally suitable for detecting a variety of Phytophthora species using pear baits.  Daily cycling of water temperature can promote zoospore release from sporangia that may be in the water.

Start checking pear baits for symptoms 2 days after the pears were added to the water.  Pears should be removed from water as soon as symptoms appear, and should be removed after 3 days even if no lesions have appeared.  Infected pears that are mostly submerged in water sometimes only develop subtle light discolored patches.  When removed from the water, the lesions will darken quickly, commonly within an hour (Figure 1).  Lesions may also appear up to 5 days (or rarely longer) after pears have been removed from the water.  

There is no advantage to leaving the pear in the water once lesions appear, as these lesions will become more vulnerable to infection by other organisms, which can complicate diagnosis.  If water chemistry is unusual (e.g., highly acid), pears may develop a network of splits or cracks, sometimes starting at wounds. Cracking also develops on some pears for other reasons that are not clear, but are likely related to the physiology of the pears. Pears should be removed from water immediately if substantial cracking develops, as they will only become more degraded if they remain in water.

Use clean waterproof gloves to remove pears from water samples and change gloves or wash them between samples.  Use soap and water or alcohol (70% isopropanol) to clean the gloves and then rinse thoroughly with water.  Rinse each pear individually with running tap water over a sink when you remove it from a sample bag.  Pears may develop a slippery biofilm on the surface; this does not have to be rinsed off completely.  Be careful to avoid cross-contamination of other samples from splashing water and handling wet pears. 

Set pears to dry on racks or clean paper towels so they do not touch each other.  Keep the pears indoors at temperatures between 18-24°C (65-75°F).  Lesions will continue to grow in size and coalesce.  For many Phytophthora species, this process will occur faster at warmer temperatures.

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Figure 1.  Pear bait from bench testing protocol showing change in symptoms appearance over time.  Top left - pear incubated in water for 3 days shows slight discoloration on the portion exposed to air.  Top right – reverse side of pear immediately after removal from water shows only very subtle discoloration.  Bottom left – 30 minutes after removal from water, infected areas have begun to darken.  Bottom right – 13.5 hours after removal from water, pear shows “classic” dark, firm Phytophthora lesions, occurring as both large consolidated lesions and small individual spots.

3.2.3.  Assessing pears for symptoms

In floating pears, lesions may develop on any portion of the pear that has been in the water (Figure 1).  Phytophthora lesions may occur either in nonwounded areas of the pears or may be associated with wounds.  They range from dark to light brown and are normally somewhat to quite firm initially, though they may become soft in the center.  Symptom intensity and the number of lesions increases with the amount of inoculum in the water.  Positive samples may have only one or two spots at low inoculum levels or may be covered with lesions under high inoculum levels.  However, Phytophthora species vary in their aggressiveness to pear baits, so the number of spots does not always correlate with inoculum level.

Lesions associated with Pythium infections or true fungi are always associated with wounds.  They normally start out soft and watersoaked and are commonly sunken.  “Classic” firm, dark brown lesions developing independent of wounds (Figure 1) are rarely, if ever, anything other than Phytophthora.  Softer, less typical lesions associated with wounds can also be caused by some Phytophthora species.  See Using green pears to bait for Phytophthora for additional images of Phytophthora infections on pear baits.

3.2.4.  Confirmation of Phytophthora on baits

Additional testing is needed to definitively confirm that lesions developing on pear baits are caused by Phytophthora species. Molecular-based Phytophthora tests may be used on pear lesions to confirm the presence of Phytophthora.  However, tests that cross-react with Pythium or Phytopythium species (e.g., Agdia test strips described above) may not provide definitive results.  Culturing and lab testing can confirm whether “non-classic” lesions are caused by Phytophthora and can be used to identify the Phytophthora species present.  Some diagnostic labs will accept pear baits, but check with the lab in advance to determine when they are willing to accept samples.  Infected pear baits are highly perishable, and if the lab cannot process them in a timely way, they may become unusable.  Pears should be sent or delivered to the processing lab as soon as symptoms are visible.  Make sure each pear is labeled.  Wrap each pear individually in several layers of clean paper towels and seal each in a labeled zip-closure plastic bag.  Carefully pack all bagged baits in a box with enough padding to prevent shifting and bruising.  Use next-day delivery if possible; two-day delivery may be acceptable if pears have only early symptoms when shipped and weather is cool.

It is also possible to identify water molds on pear lesions as Phytophthora based on the method of zoospore release. This requires a compound microscope and training in how to recognize and differentiate Phytophthora from Pythium or Phytopythium.  Using sterilized tweezers, take small pieces (a few square mm) of pear epidermis from the edge of the lesion(s) of interest and float them in a small amount of water (a few mm deep) in a petri plate. Use non-chlorinated water, such as distilled or carbon-filtered water. Mycelium will start to grow out from the pear pieces into the water in about a day. A few sporangia or other spores (such as oospores) may appear within a day, but usually 2 more days are required for substantial numbers of sporangia to form. If no sporangia form within 3 days, replace the water in the petri plate with fresh water. To induce zoospore release in mature sporangia, chill the plates for about 20 minutes in a refrigerator, then allow the plates to rewarm to room temperature. After chilling, check the plates under a microscope at about 30 minute intervals to look for sporangia that are releasing zoospores. In Phytophthora species, zoospores fully differentiate within the sporangia. They begin swimming immediately as they exit the sporangium or may push out of the sporangium in a blob from which zoospores quickly disperse (within a few seconds). In contrast, zoospores of Pythium and Phytopythium differentiate in a globose vesicle that is pushed out of the sporangium.

3.2.5.  Disposing of infected baits

Pear baits that are used for baiting should be heat-treated to kill Phytophthora before disposal.  Heating pears in a microwave in a heat-resistant plastic bag to a temperature of 95 C (203 F) for 30 seconds is sufficient to kill these pathogens in infected pears.  An alternative standard is 85 C (185 F) for 3 minutes.  Allow the bag to cool slowly to maximize the duration of the high temperature treatment.

3.2.6. Other baiting methods

Baiting with green pears can also be used to detect Phytophthora in root/soil samples or in water collected from water bodies, watercourses, or runoff. See Using green pears to bait for Phytophthora for details on these techniques and more photos of pear symptoms.