Phytosphere Research

Using green pears to bait for Phytophthora

See also Testing Procedures for BMPs for Producing Clean Nursery Stock

Elizabeth Bernhardt and Ted Swiecki, Phytosphere Research

Because Phytophthora species can be difficult to isolate from diseased plants, plant pathologists sometimes use trap plants, or baits, to detect Phytophthora. A review of baits used can be found in 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 Phytophthora species, and are relatively easy to interpret. Pears can be used to detect Phytophthora in soil samples, water samples, and root samples.


Handling different sample types

Water samples: pear baits should be placed in water samples when they are collected because actively swimming zoospores are likely to be present if the sample is infested. If zoospores encyst before baits are added, baiting may provide a false negative result. Most pears will float in water. If the pear sinks, 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.

Soil / root samples: If samples are from a moist site (e.g., irrigated nursery stock, field site with moist soil near field capacity), pears can be introduced directly into the sample and the sample can be flooded immediately as described below. If soil has been dry for an extended period, detection of Phytophthora can be enhanced by adding enough water (use carbon filtered tap water, tap water which has sat for 24 hours to allow chlorine to dissipate, bottled drinking water, or distilled water) to bring soil to about field capacity (moist but not at all saturated). Allow the sample to sit for 48 to 72 hours before adding pears and additional water as described below.


Selecting and preparing pears

It is important that pears be as green and as unwounded as possible. Phytophthora species are among the few organisms that can infected green, unwounded pears. Ripe pears and those with nicks and scratches are susceptible to invasion by organisms other than Phytophthora. Smooth, green-skinned varieties (e.g., Bartlett, D' Anjou, Packham) are all acceptable.

Rinse pears before using or wash with a small amount of dish soap and rinse well. Using a permanent marker, number the pear with the sample number near the sem 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 what you see when pears are removed after baiting. You will want to keep any small wounds above the water level (not possible for water baiting). Don't try to peel off the grocery sticker, as it will generally tear the pear skin. If possible, leave it above the water line as well, otherwise, don't worry about it.


Baiting procedures

Samples to be baited can be placed in clean sturdy plastic bags or containers as shown below. Containers should be new or cleaned and rinsed well so that no detergent residue is present. Bags simplify cleanup, but need to be placed in containers to contain leaks (if an undetected hole is present) and to support the sample to keep the pear in position. Phytophthora zoospores have been reported to swim up to about 7 cm through still water, and smaller distances through saturated soil, so there is no advantage to using overly large containers or samples.

Pears can be placed upright or tilted at about a 45 degree angle, which increases the contact area with the water somewhat. If tilted, keep the stem above water. If the sample includes hard particles (e.g., sand grains, perlite, rock fragments), you need to insert the pear carefully to avoid wounding it when it is placed in the sample. If using a plastic bag, you can use your hand from the outside of the bag to create a depression where the pear will rest. Place the pear so that any wounds you may have detected will be above the water line. In a typical soil/root sample, about 2 to 3 cm of the pear will rest in the depression in the sample. Flood the sample with water so that the water line will be about 1 to 2 cm above the soil level.

Incubation temperatures with diurnal fluctuations from 21C to 27C are generally suitable for detecting a variety of Phytophthora species using pear baits. Phytophthora species prefer well-aerated conditions, so leave bags open on top to allow for air exchange.

Baiting a large soil / root sample in a plastic bin. Two baits are used to increase the detection efficiency in this large sample.

Baiting a large number of small samples using 1 quart plastic bags

pear baits Overhead view of pear baits in 1 gal bags.


Assessing baits

The reaction on the pears will vary depending on the amount of inoculum in the soil. Often a "bathtub ring " of spots will form at the water line, as in the images below. Infections at the water line are commonly initiated by zoospores released from sporangia on root fragments in the soil. Zoospores tend to swim toward the water surface and are also attracted to host materials including roots, fruits such as pears, leaves of some species, and some kinds of seedlings.

"Bathtub ring" of Phytophthora infections that were initiated near the water line of pear baits. Lower image is of a pear that was tilted during baiting.

Start checking the pear for symptoms after 2 to 3 days. If necessary, carefully pick up the pear and check the bottom, being careful to contain all drips in the sample bag, and gently replace the pear after inspection if it lacks obvious symptoms. It may be helpful to gently redistribute the soil sample somewhat at this point. The objective is to free up or redistribute sporangia that may be trapped at the bottom or corner of the bag or or container. Be careful to avoid cross contamination between samples when handling pears. If possible use gloves and wash hands/gloves between samples with soap and water or rinse with alcohol (70% isopropanol) and then water (to avoid dripping alcohol into the samples).

Remove pears after 5 days, even if no spots have shown up yet. In some situations, lesions may become visible a day or two after the pear has been removed from the flooded sample.

Once lesions become visible, remove the pear and rinse it well with tap water at a sink. Be careful to avoid cross-contamination of other samples from splashing water. There is no advantage to leaving the pear in the water once lesions appear, as these necrotic areas will become more vulnerable to infection by other organisms, complicating diagnosis. Set pears to dry on racks or paper towels so they do not touch each other. You can keep them at the same temperature range used for baiting. Spots will continue to grow in size and coalesce. In general, this process will occur faster at warm temperatures. The pears in the image below were all removed from a series of soil samples at the same time, and show strong differences in infection due to variable amounts of inoculum in the soil.

Pears exposed to varying levels of Phytophthora inoculum in soil samples. Top two rows show decreasing levels of inoculum from left to right. The three pears at the bottom were incubated in soil samples that were negative for the presence of Phytophthora. Phytophthora cinnamomi was isolated from lesions on all the pears in the top two rows in the image above. Note the dotted lines on some pears, which encircle various wounds or imperfections seen before baiting.

Phytophthora forms dark brown, initially firm lesions on pears. The lesions will become softer over time and can develop a water soaked appearance near the edges. Much of the submerged portion of the pear bait may be covered with brown lesions within 2 to 3 days after being placed in the flooded sample if the sample has high amounts of Phytophthora inoculum. These lesions can merge quickly, so the affected part of the pear may turn completely brown. If the amount of Phytophthora inoculum is low, isolated spots may form, often at the water line or on the bottom of the pear where it was in contact with the soil or roots.

Observations of brown lesions on unblemished green pears strongly suggest that the sample contains Phytophthora species. Lesions that start out soft and watersoaked are commonly associated with Pythium infections or various fungi. Although "bathtub rings" of lesions are almost always due to Phytophthora infections, individual spots are more difficult to interpret. The appearance of the lesion can be a good indicator, but is not definitive. To confirm the pears have been infected by a Phytophthora species, it is necessary to confirm the presence of the pathogen through further testing.

Upper photo, spots caused by Phytophthora cinnamomi, lower photo, spots caused by Pythium species.


In above photo, spots on left and right pear are caused by different Phytophthora species - lesions on both pears are firm.


In above photo, spots are caused by another Phytophthora species at two different zoospore concentrations.


Confirmation of Phytophthora infection in baits

Phytophthora infections in pear baits can be confirmed though several methods.

Direct observation: Pieces of lesions floated in water in petri plates will commonly form sporangia, which can be identified as Phytophthora based on the method of zoospore release. This requires a microscope and training in how to recognize and differentiate Phytophthora from similar microorganisms.

Isolation on media: To isolate Phytophthora from baits onto media, pears are first disinfested by placing them in 0.5% NaOCl (diluted bleach) for 30 to 60 seconds. Place the pear on a clean paper towel and let the bleach solution dry off a bit (you can also rinse it off with sterile water).

Cut out small pieces from the edges of suspect lesions and place them into agar media in petri dishes. Use aseptic technique throughout, and sterilize implements and keep the media from becoming contaminated. Be sure to flame your tools before starting and after each piece is transferred to the media. Pieces should be small, no more than about 3mm square and should be slipped into the agar below the surface. Various media can be used, including selective media such as PARP agar or general media such as cornmeal agar (CMA).

Petri dishes should be examined daily. In general, Phytophthora species grow relatively slowly, increasing in diameter by up to about 1 cm per day. If faster growing colonies appear on the plate, use sterile technique to cut small pieces from the edges of slower growing colonies and transfer them to new plates before they are overrun, which will make it impossible to confirm that Phytophthora is present. Mycelium growing out of the tissue pieces should be examined periodically with a microscope. Phytophthora species usually have a characteristic appearance that helps to distinguish them in culture from other genera of water molds and true fungi. A few species can be identified with a high degree of certainty due to their characteristic appearance in culture, but genetic sequencing is generally required to determine the identity of Phytophthora species.


Negative results

It must be understood that baiting, like every other test for the presence of Phytophthora, is subject to false negative results. Negative results may result for a variety of reasons, so a negative result in a single test is not conclusive. Plants treated with fungicidal materials such as phosphites or mefenoxam may yield negative results but still contain viable (though suppressed) Phytophthora infections. Repeated samples from a given infested site can yield both positive and negative results, especially if inoculum levels are moderate to low or infections are distributed nonuniformly. If repeated testing under a range of conditions conducive for Phytophthora detection consistently provides negative results, it is more likely that negative results are actually meaningful, indicating that Phytophthora is either absent or present in levels that are below the detection threshold. In contrast to negative results, recovery of Phytophthora through baiting is conclusive evidence that the pathogen was present in the sample


Reference

Erwin, D. C., and Ribeiro, O. K. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society Press, St. Paul, MN.

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