The authors gratefully acknowledge funding from the US Forest Service Forest Health Protection Evaluation Monitoring and Special Technology Development Programs that have supported the development, modification and application of the protocol described here to identify trees that are resistant and susceptible to the beech scale insect.&#160;We also are grateful to the Holden Arboretum, Kirtland, OH, for their continued partnership and support of our American beech resistance screening program.

1. Plant Material: Mature Field Trees, Potted Seedlings, or Potted Grafts
<ol>
	<li>For field testing, select mature healthy American beech trees that show no signs of scale infestation or disease for testing for possible resistance. Visibly susceptible trees will also need to be identified to be used as a control (Figure 1).</li>
	<li>For testing potted seedlings or grafts, collect and germinate beechnuts as described in Koch &#38; Carey, 2004 or graft scion as described in Carey et al, 2013.</li>
	<li>Grow seedlings or grafts in potting soil mix amended with 47 g micronutrients, 477 g slow release fertilizer 15N-3.9P-9.9K, 700 g coarse perlite and 75 g aluminum sulfate per 2.8 cu. ft. bag. If needed later in the growing season, fertilize plants weekly with soluble 17N-1.3P-14.1K at 200 ppm nitrogen.</li>
	<li>During the growing season, keep plants in a shade house. Allow plants to go dormant outside in the fall prior to moving them to a controlled temperature storage facility (~4 &#176;C) from November through April.</li>
</ol>
2. Collection of Beech Scale Eggs
<ol>
	<li>In a BBD-infested stand, inspect heavily infested trees (easily identified by their &#8220;whitewashed&#8221; appearance, Figure 1A) with a hand magnifying glass to confirm the presence of eggs, typically abundant from mid-July to mid-August.</li>
	<li>Use a paintbrush to gently brush the white waxy clumps of adult scale insects, eggs and other debris, into a plastic sealable one gallon collection bag (Figure 1B). Collect from a minimum of three different trees at least 12 m apart.</li>
	<li>If needed, store eggs in the sealed collection bag for up to two weeks at 4 &#176;C. Tape a small piece (2.5 cm square) of dampened polyethylene foam to the inside of the bag to prevent the eggs from drying out.</li>
	<li>To separate the scale eggs (0.15 x 0.25 mm) from the adults (0.60 mm) and debris, construct a sieve using a short piece of 2&#8217; PVC pipe and a coupling to support a square piece of 250 micron nylon mesh (Figure 2).</li>
	<li>Empty the mixture of adults, wax, eggs, and debris from the collection bag onto the sieve and use a small paint brush to gently encourage the eggs to pass through the mesh into a glass Petri dish below. Plastic Petri dishes&#160;should be avoided because they hold more static electricity, making it difficult to move the eggs. Eggs before and after sieving are shown in Figure 3.</li>
	<li>Purified eggs can be stored in the Petri dish at 4 &#176;C for at least a week. To prevent the eggs from drying out, tape a damp piece of foam to the lid and seal with Parafilm.</li>
</ol>
3. Egg Viability Assays
<ol>
	<li>To assess egg viability, use a 10 cc syringe to apply a thin ring of petroleum jelly around the circumference of the bottom of a 60 mm glass Petri dish (Figure 4A).</li>
	<li>Transfer about 100 eggs to the center of the ring, place the lid on the Petri dish and seal with Parafilm. Allow the sealed Petri dish to remain undisturbed at room temperature for 2 weeks, or 3 weeks if eggs were stored at 4 &#176;C prior to beginning the assay.</li>
	<li>Hatched nymphs will become stuck in the petroleum jelly and can be easily counted, and empty eggs are easily distinguished from unhatched eggs by their color and sheen (Figure 4B). Calculate viability by dividing the number of nymphs by the sum of the empty eggs plus the remaining full eggs. Good viability should be in the 75% to 90% hatched eggs range.</li>
</ol>
4. Scale Resistance Screening of Large Mature Beech Trees in the Field
<ol>
	<li>For a quantitative test, count out 500 eggs using a dissecting microscope and using a small spatula gently sprinkle them across the center of a predampened, open-cell 10 x 15 x 1.3 cm rectangle of polyethylene foam. To dampen foam, wet it then squeeze out as much water as possible. For a qualitative test, 500 eggs can be counted out and placed in a small glass vial and a &#8220;fill&#8221; line drawn that can be used to measure additional batches of approximately 500 eggs.</li>
	<li>Place the foam pad onto the test tree with the egg side facing against the bark. Hold the pad in place with rope, string, twine, or plastic coated hardware wire. Plastic or metal based material should be used rather than natural fiber materials which are more readily scavenged by wildlife.</li>
	<li>Line the top and sides of a precut 23 x 30 cm piece of vapor permeable waterproof house wrap with acetate based silicone adhesive and place it over the foam test pad. Press the edges of the house wrap to the tree to create a waterproof seal. Place nylon twine or plastic coated hardware wire around the tree and house wrap, to hold it in place while the adhesive sets (Figure 5).</li>
	<li>Place a minimum of two test pads on each test tree, preferably on opposite sides of the bole. At each site, place test pads on at least 2&#160;susceptible trees (with obvious natural scale infestation) as a control. Prior to placing the test pads on control trees, remove any naturally occurring scale insects or eggs using a firm bristled brush. 
	Note: Foam pads with eggs can also be placed on trees for the purpose of rearing scale eggs, which can be particularly helpful in areas where the infestation level is low.</li>
</ol>
5. Scale Resistance Screening of Potted Seedlings or Grafts
<ol>
	<li>For testing, select potted trees with a minimum diameter of 1 cm (caliper 5 cm above soil line) that are tall enough to have at least 2&#160;separate test pads placed on them. Prune small side branches when necessary to make room for the test pads.</li>
	<li>Use a dissecting microscope to count out 150 eggs and scatter them over a precut and dampened 2.5 x 7.6 x 1.3 cm open-cell polyethylene foam pad.</li>
	<li>Affix the foam pad to seedling with the egg side against the bark, using plastic-coated wire. Wrap a small square of house wrap around the seedling just above the foam and seal it with acetate-based silicone (see Figure 6A).</li>
	<li>Include known susceptible seedling families or grafts as controls.</li>
</ol>
6. Data Collection
Note: Approximately 52 to 57 weeks after placing the foam pads and eggs on the test trees, data can be collected. It is important that this is not done until after adults have begun to lay eggs so that their ability to reproduce can be determined. On some resistant trees it is not unusual to see a small number of adults establish but with no reproduction occurring.
<ol>
	<li>Carefully remove the foam pad and count the number of adult scale insects established on the bark using a hand lens or magnifying glass (10X).</li>
	<li>When the foam pad is removed, it is not uncommon for some adults and most egg clusters to be pulled off of the tree with the foam (see Figure 7). Using a dissecting microscope, count the egg clusters and adults that have remained attached to the foam pad.</li>
</ol>

<ol>
	<li>Houston, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+artificially+infest+beech+bark+with+the+scale,+Cryptococcus+fagisuga+(Lindinger).">A technique to artificially infest beech bark with the scale, Cryptococcus fagisuga (Lindinger).</a> U.S. Forest Service. , (1982).</li><li>Houston, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+beech+resistance+to+Cryptococcus+fagisuga.">American beech resistance to Cryptococcus fagisuga.</a> Proceeding, IUFRO beech bark disease working party conference. , 38-42 (1983).</li><li>Ehrlich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beech+bark+disease:+a+nectria+disease+of+Fagus,+following+Cryptococcus+fagi.">The beech bark disease: a nectria disease of Fagus, following Cryptococcus fagi.</a> 10, 593-692 (1934).</li><li>Papaik, M. J., Canham, C. D., Latty, E. F., Woods, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+an+introduced+pathogen+on+resistance+to+natural+disturbance:+beech+bark+disease+and+withdrow.">Effects of an introduced pathogen on resistance to natural disturbance: beech bark disease and withdrow.</a> 35, 1832-1843 (2005).</li><li>Miller-Weeks, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+beech+bark+disease+in+New+England+and+New+York.">Current status of beech bark disease in New England and New York.</a> Proceeding, IUFRO beech bark disease working party conference. , 21-23 (1983).</li><li>Houston, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beech+bark+disease:+The+aftermath+forests+are+structured+for+a+new+outbreak.">Beech bark disease: The aftermath forests are structured for a new outbreak.</a> J. For. 73, 660-663 (1975).</li><li>Twery, M. J., Patterson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+beech+bark+disease+and+its+effects+on+species+composition+and+structure+of+northern+hardwood+stands+in+central+New.">Variations in beech bark disease and its effects on species composition and structure of northern hardwood stands in central New.</a> 14, 565-574 (1984).</li><li>Jakubus, W. J., McLaughlin, C. R., Jensen, P. G., McNulty, S. A., Evans, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternate+year+beechnut+production+and+its+influence+on+bear+and+marten+populations.">Alternate year beechnut production and its influence on bear and marten populations.</a> Beech Bark Disease: Proceedings of the Beech Bark Disease Symposium. , 79-87 (2005).</li><li>Koch, J. L., Carey, D. W., Mason, M. E., Nelson, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+beech+scale+resistance+in+full-+and+half-sib+families.">Assessment of beech scale resistance in full- and half-sib families.</a> Can. J. For. Res. 40, 265-272 (2010).</li><li>Koch, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beech+bark+disease:+The+oldest+&#8220;new&#8221;+threat+to+American+beech+in+the+United+States.">Beech bark disease: The oldest &#8220;new&#8221; threat to American beech in the United States.</a> Outlooks Pest Manag. 21, 64-68 (2010).</li><li>Koch, J. L., Mason, M. E., Carey, D. W., Palmieri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+resistance+to+beech+bark+disease:+improvements+and+results+from+seedlings+and+grafted+field+selections.">Screening for resistance to beech bark disease: improvements and results from seedlings and grafted field selections.</a> Proceedings of the 4th International Workshop on Genetics of Host-Parasite Interactions in Forestry. , (2012).</li><li>Leak, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fifty+year+impacts+of+the+beech+bark+disease+in+the+Bartlett+Experimental+Forest,+New+Hampshire.">Fifty year impacts of the beech bark disease in the Bartlett Experimental Forest, New Hampshire.</a> Northern J. Appl. For. 23, 141-143 (2006).</li><li>Koch, J. L., Carey, D. W., Yaussy, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cross-pollinations+with+American+beech+trees+that+are+resistant+to+beech+bark+disease.">Controlled cross-pollinations with American beech trees that are resistant to beech bark disease.</a> Proceedings of the 14th Central Hardwood Forest Conference. , 358-364 (2004).</li><li>Carey, D. W., Mason, M. E., Bloese, P., Koch, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+callusing+for+propagation+of+American+beech+by+grafting.">Hot callusing for propagation of American beech by grafting.</a> HortSci. 48, 620-624 (2013).</li><li>Koch, J. L., Carey, W. W., Evans, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+resistance+of+American+beech+to+beech+bark+disease:+knowledge+through+2004.">The genetics of resistance of American beech to beech bark disease: knowledge through 2004.</a> Beech Bark Disease: Proceedings of the Beech Bark Disease Symposium. , 98-105 (2005).</li></ol>

The authors declare that they have no competing financial interests.

The critical steps required for success of this assay include performing a viability test on the eggs and the use of susceptible controls at each site tested, and with potted materials.&#160;It is also important to use more than one test pad per tree or seedling.&#160;We have found that both in the field and in more controlled tests on potted plants, a frequent source of error can be pad failure.&#160;For example, in the field, even with our modification of covering the test pad with house wrap to prevent excess moisture...

Beech bark disease (BBD) has had a detrimental impact on American beech in North America since the introduction of the invasive beech scale insect, Cryptococcus fagisuga, in the Canadian province of Nova Scotia in the late 1890s3.&#160;This insect-disease complex is initiated when the beech scale insect inserts its feeding stylet into the bark creating small fissures that provide entryway for infection by one of the Neonectria species of fungus (Neonectria ditissima or Neonectria faginata).&#160;As the fungal mycelia grow, large areas of tissue may die, eventually completely girdling the tree.&#160;The damage from the disease weakens the tree, making it prone to snapping in high winds4.&#160;Mortality levels in the first wave of the disease have been reported to be as high as 50%5.&#160;Surviving trees are often severely deformed as cankers form reducing value of the tree as a wood product.&#160;Such trees have a propensity for root-sprouting which leads to the formation of &#8220;beech thickets&#8221; that prevent other more desirable species from establishing,&#160;reducing&#160;the economic and ecological value of the stand6.&#160;Although beech bark disease is not likely to lead to extinction of the American beech, it alters stand composition and health leading to a decrease in food and habitat for wildlife7,8.

In stands affected by BBD for many years, trees that remain free of any symptoms of the disease have been reported.&#160;Artificial inoculation trials have shown that these trees are resistant to the scale insect2.&#160;Without scale infestation, there is little opportunity for Neonectria infection, minimizing the impact of the fungus.&#160;Large scale mortality in American beech due to Neonectria infection in the absence of prior scale infestation has never been reported, so resistance to the beech scale insect results in resistance to BBD.&#160;

Recent research on management of BBD has focused on the identification, propagation, breeding, and retention of American beech trees with resistance to the beech scale insect.&#160;Genetic studies have shown that resistance to the scale insect is heritable and careful selection and breeding of resistant trees can result in significant improvement in a single generation9.&#160;This finding has fueled efforts by state and national forest managers in the United States to establish regional seed orchards of resistant American beech to provide a source of genetically diverse BBD-resistant seed for restoration plantings10,11. Research has also indicated that silvicultural manipulation of stand genetics by the removal of susceptible trees and retention of resistant trees can result in stand improvement9,12.&#160;

Management of BBD through tree improvement activities or through carrying out silvicultural prescriptions requires the ability to select for and distinguish between beech scale-resistant and susceptible trees.&#160;The methods presented here have been adapted from a method first introduced by Dave Houston to artificially inoculate seedlings with beech scale eggs1.&#160;The method can be used as a screening tool to identify quantitative trait loci (QTL) associated with resistance or to distinguish between resistant and susceptible potted seedlings or grafted ramets in genetic studies.&#160;Alternatively, it can be used for screening mature trees in the field to identify resistant trees for seed orchard development, or retention in the field.&#160;Susceptible trees can be identified and removed to minimize disease impacts.&#160;

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a technique to screen american beech for resistance to the beech scale insect (cryptococcus fagisuga lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

Figure 6 shows a resistant seedling (C) and two susceptible seedlings (D, E) exhibiting different degrees of susceptibility. An example of what a susceptible mature tree looks like 57 weeks after the artificial infestation test was set up is shown in Figure 5B. When the foam is peeled away from the tree, it is not uncommon for scale insects and their egg clusters to remain stuck to the foam as shown in Figure 7, which is...

Watch this Scientific Journal Video about A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind. at JoVE.com

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

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9.5K Views.  US Forest Service. Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark. Trees that are resistant to the scale insect are also disease resistant. Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.