Version 12/20/2019
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). Baiting relies on the fact that Phytophthora zoospores are chemotactically attracted to host materials including seedlings, roots, fruits, and leaves and actively swim toward these materials following chemical gradients of plant exudates. Different baits vary in their ability to attract and become infected by the various Phytophthora species, and no single bait can detect all species.
Among baits that have been identified, green pears are susceptible to a wide variety of common and uncommon Phytophthora species. More than 50 taxa of Phytophthora, including previously undescribed species and apparent hybrid species have been detected using pear baits. Green pears are also readily available (all year in California), are relatively easy to interpret, and can be processed without intensive lab work. Baiting only detects living Phytophthora propagules, so false positive results are not possible if bait infections are confirmed by culturing. A false negative result from baiting with pears, i.e., a lack of detection when viable Phytophthora is actually present, may result if the Phytophthora species present does not readily infect pears, or there is not enough inoculum present to infect the pear.
Baiting primarily detects zoospores present (in water samples) or released from sporangia (in soil/root samples). Some infections of pear baits in soil/root samples may also originate from other spore types or mycelia where the bait is in contact with infested soil or infected roots. Sample handling and processing are critical to obtaining good detection efficiency by baiting, especially if inoculum levels in the sample are low. Conditions need to be favorable for sporangium production, zoospore release, and bait infection. Suboptimal conditions can result in false negative results.
Zoospores tend to swim upward toward the water surface in opposition to the force of gravity (negative geotaxis). Hence baits should to be at or near the water surface. A Phytophthora zoospore will generally swim no more than 8 to 24 hours, with shorter swimming duration at higher temperatures generally. Mechanical agitation and rapid changes in temperature or the salt concentration of the water can induce zoospores to encyst, so these impacts should be avoided during sample transport and the baiting process.
Baiting with green pears can be used to detect Phytophthora in root/soil samples or in water collected from water bodies, watercourses, runoff, and irrigation leachate. This document presents information on baiting water samples, including irrigation leachate samples. See this link for additional information about using pears to bait soil/root samples.
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. Therefore, it is important that pears selected as baits be as green and as free of wounds and blemishes as possible. Don’t use pears with large wounds and avoid pears with many wounds, especially fresh wounds. Also avoid pears with extensive russeting or blush. Smooth, green-skinned varieties (e.g., Bartlett, D' Anjou, Packham) are all acceptable, but D’Anjou is preferable because this variety ripens more slowly than the other varieties. It is not necessary to use organically-grown pears.
Before use, wash pears with a small amount of diluted dish detergent and rinse well to remove all detergent residues. Don't try to peel off the grocery sticker, as this will generally tear the pear epidermis. Using a permanent marker, number the pear with the sample number near the stem end. If there are small surface wounds or discoloration, you can use the marker to make a light dotted line around the affected areas, which can help you interpret symptoms that are visible on pears during and after the baiting period. For root/soil samples, position pears to keep any small wounds above the water level. Because this is not possible for water samples, try to avoid using pears with obvious wounds.
For water samples, the pear bait should float at the surface to maximize infection efficiency. Most pears will float in water, though this is variable between batches of pears depending on how long they have been in storage. After pear baits are washed as noted above, test them individually in a container of clean water and set aside or mark any that any do not float. Pears that sink can be used for root/soil samples (pears rest on the soil sample and don’t need to float) or can be provided with a pear floatation device (PFD). A simple PFD consists of a small piece of closed cell foam (e.g., a packing peanut but not the water-soluble type) that is attached to the pear with a clean rubber band. Orient the rubber band to encircle the long axis of the pear.
Figure 1. Pear with pear flotation device (PFD).
Baits need to be added to samples when actively swimming zoospores are likely to be present (see description of the disease cycle of Phytophthora root rots). For this reason, the bait should be added to the sample as soon as zoospores are likely to be present in the specific test procedure being used. For water samples, pears are added at the time the water is collected. The bench leachate protocol used to detect Phytophthora in container nursery stock is a specialized type of water sample. Pears are added to the vessels used to collect the leachate before the test is started.
Do not subject baited water samples to sudden changes in temperature or mechanical agitation. If zoospores encyst before infection of the pear bait can occur, baiting may provide a false negative result. Zoospore motility is generally prolonged at cooler temperatures, but temperature optima differ among Phytophthora species. To provide favorable conditions for a range of Phytophthora, transport and maintain baited water samples where they can gradually equilibrate to a moderate temperature (about 18-24°C [65-75°F]).
We normally collect water samples in heavy duty 1-gallon zip-closure bags (e.g., 1-gallon Freezer Ziploc bags). These bags can comfortably hold up to about 2.7 L of water plus a pear. When water is added to these bags, they need to be supported in a container to prevent the sides from collapsing and spilling the water. For water samples, up to about 2.7 L of water in a 1-gallon bag can be supported in a 1-gal plastic bottle (about 15-16 cm diameter) cut off to a height of about 17 cm. The supporting container should be large enough to prevent any accidental spills or leakage from the bag. It is a good idea to place these containers on a tray or in a shallow bin that can provide secondary containment of spills or leakage. During incubation, check bags for leaks, especially in the first day or two. If a substantial leak is detected, place the sample bag into a second bag.
If samples need to be transported, bags should be zipped closed to prevent spills and cross-contamination. Include a large air bubble at the top of the bag when it is sealed and keep the baited samples in an insulated cooler (without ice) to minimize the rate of change in water temperature. When samples are brought to where they will be incubated, open the sample bags.
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 suitable for detecting a wide variety of Phytophthora species using pear baits. Daily cycling of water temperature can promote zoospore release from sporangia that may be in the water. Zoospore cysts of some Phytophthora species can release a secondary zoospore via a process known as repeated emergence, which may also be favored by some variation in temperature. You can incubate samples in a room with the appropriate temperature regime or place them in an incubator. A simple indoor incubator can be built at low cost with a few easily acquired parts.
Phytophthora symptoms on pear baits usually begin to show three or more days after initial exposure to zoospores, though symptoms may show up a bit earlier. Start checking baits for symptoms after two full days of incubation and continue to check daily until the end of the flooding period. Pears should be removed from water as soon as definite Phytophthora symptoms appear. Secondary organisms such as Pythium species may begin to colonize the lesions if they remain in water, which can complicate diagnosis. 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 2).
For water samples, including irrigation leachate, standard practice is to remove pears from the water after 3 full days even if no lesions have appeared. This period can be extended for up to a day if pears are still in good condition, but longer periods of incubation in water are not likely to improve sensitivity and pears tend to develop more issues from prolonged water contact. After removal, pears should be observed for an additional 5 days for the appearance of lesions that may develop late or slowly. Hence, a standard test requires at least 8 days from the start of baiting before a negative result (no detection) is recorded.
If water chemistry is unusual (e.g., highly acidic), pears may develop a network of splits or cracks, sometimes starting at wounds. Cracking also develops on some pears for other reasons that are probably related to the postharvest 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.
Be careful to avoid cross-contamination between samples from splashing water and handling wet pears. Use clean waterproof gloves to remove pears from water or soil/root sample bags. Rinse each pear individually with cool tap water when you remove it from a sample bag to remove surface debris. Pears may develop a slippery biofilm on the surface; this does not have to be rinsed off completely. To prevent cross contamination between samples, clean your gloves thoroughly with soap and water or alcohol (70% isopropanol) and then rinse them thoroughly with water to remove residual soap or alcohol.
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). Inspect the pears once or twice a day to check for lesion development.
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Figure 2. Pear bait from bench leachate testing protocol showing the rapid change in coloration that can develop when pear baits are removed from the water sample. 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.
See 3.3. Individual plant sampling/baiting and 3.4. Bench testing leachate from batches of container nursery plants for additional details on on how to use pear baiting for testing nursery stock.
In floating pears, lesions may develop on any portion of the pear that has been in the water (Figure 2). Phytophthora lesions can develop in nonwounded areas of the pears, but they can also be associated with wounds. Phytophthora lesions can develop in nonwounded areas of the pears, but they can also be associated with wounds. Phytophthora lesions range from dark to light brown and are normally somewhat to quite firm initially because they are largely confined to the pear epidermis. Over time, the older portions of the lesions may become softer and slightly sunken. Lesions caused by some Phytophthora species may have a somewhat water-soaked appearance at the edges. “Classic” firm, dark brown lesions that develop in nonwounded tissue (Figure 2) are rarely, if ever, anything other than Phytophthora. However, some softer, less typical lesions associated with wounds can also be caused by some Phytophthora species.
Pears may also develop lesions associated with Pythium infections or true fungi. . These are always associated with wounds. They normally start out soft and watersoaked and are commonly sunken. Pythium lesions also tend to have a translucent appearance because decay extends into the fleshy portion of the fruit. Lesions caused by Phytophthora and Pythium will continue to grow in size and coalesce. This process will generally occur faster at warmer temperatures. Pythium lesions can expand rapidly, and may overrun small Phytophthora lesions. Lesions caused by abiotic factors or by some true fungi may become limited and stop expanding over time, though the tissue may collapse as it dehydrates.
In some instances, water or soil chemistry can cause surface discoloration of the pear epidermis that will be limited to the contact area. This discoloration can be distinguished from a Phytophthora lesion in that it will not expand further once the pear is removed from the water, whereas Phytophthora lesions will continue to expand. These uncommon reactions of pear baits may interfere with the test, especially if they are severe and develop within the first two days of incubation. If test is negative or difficult to assess due to such issues, conduct a new test if possible.
See Using green pears to bait for Phytophthora/Interpreting symptoms on baits for additional images of Phytophthora infections on pear baits and information on how to confirm whether lesions are caused by Phytophthora.Pear baits that are used for baiting should be heat-treated to kill Phytophthora before disposal. Heating pears in a microwave in to a temperature of 95 C (203 F) for 30 seconds will kill these pathogens in infected pears. An alternative standard is 85 C (185 F) for 3 minutes (Runia and Amsing 2001). Place pears in a heat-resistant plastic bag before microwaving. The heavy duty zip-closure plastic bags used for the sample will work for this purpose. To allow steam to escape, do not completely zip seal the bag before microwaving. Place the bags in a microwave-safe container during heating to contain any liquid that may leak. Allow bags of microwaved pears to cool slowly to maximize the duration of the high temperature treatment.
Erwin, D. C., and Ribeiro, O. K. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society Press, St. Paul, MN
Runia, W. T.; Amsing, J. J. 2001. Disinfection of recirculation water from closed cultivation systems by heat treatment. Acta Horticulturae 548: 215–222.
Update 12/20/2019 - substantially edited to removed many redundancies between this page and Using green pears to bait for Phytophthora in soil root samples. Some redundancies between the two pages remain.
