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.

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

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of different gall types and prevents the development of normal shoots. Repeated and uncontrolled attacks cause, besides the production of galls and the attendant gall-related reduction in leaf area, progressive corruption of the branch architecture, including the death of branch parts, and an increase in dormant bud activation. Thus far, there have been few attempts to quantify branch architecture damage. Further, the different methods for assessing infestation degree (MAID) that have been developed focus only on the galls' presence and abundance.
Using the leaf area to sapwood area relationship as a green biomass indicator, we developed in a previous study a damage composite index (DCI) that takes into account the most important branch architectural features, allowing for realistic damage assessment during the entire epidemic process. 
The aim of this study is to present this novel method and highlight differences in the damage description with respect to other broadly used indices. Results show how the DCI depicts branch damage better, especially during the epidemic peak, compared to MAID, which tend to underestimate it. We conclude by suggesting how to properly evaluate the overall impact of the pest by means of our composite damage index, the infestation degree using classic methods, and crown transparency evaluations.

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

It is common practice to assess the damage caused by Dryocosmus kuriphilus by considering the abundance of galls alone rather than by also taking related branch corruption into consideration. We propose a composite damage index that takes into account the most important branch features, thus enabling more realistic damage assessment.

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of ...

A Method for Quantifying Foliage-Dwelling Arthropods

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density requires a method with high detection probability and a known sampling area. While most described methods provide a qualitative or semi-quantitative estimate adequate for describing species presence, richness, and diversity, few provide an adequately consistent detection probability and known or consistent sampling areas to provide an index or estimate with adequate precision to detect differences in abundance across environmental, spatial, or temporal variables. We describe how to quantify leaf-dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate and quantify them. As we demonstrate, this method can be used at a landscape scale to quantify leaf-dwelling arthropods with adequate precision to test for and describe how spatial, temporal, environmental, and ecological variables influence arthropod richness and abundance. This method allowed us to detect differences in density, richness, and diversity of leaf-dwelling arthropods among 5 genera of trees commonly found in southeastern deciduous forests.

We describe how to quantify leaf dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate for quantification.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density ...

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these insecticides against adult insects or their impact on reproductive output. However, currently available methods assess adult mortality only, limiting the selection of novel insecticides with a different mode of action. In this work, different experimental methods were examined to identify the diverse effects on the CBB under laboratory conditions. For this, green coffee fruits (GFs) were collected and disinfected by immersion in sodium hypochlorite solution followed by UV light irradiation. In parallel, CBB adults from a colony were disinfected by immersion in sodium hypochlorite solution. To assess fruit protection (preinfestation), the fruits were placed in plastic boxes, and the insecticides were applied. Then, the CBB adults were released at a rate of two CBBs per GF. The GFs were left under controlled conditions to evaluate CBB infestation and survival after 1, 7, 15, and 21 days. To evaluate insecticide efficacy after CBB infestation (postinfestation), CBB adults were released to the GFs in a 2:1 ratio for 3 h at 21 &#176;C. Infested fruits showing CBB adults with their abdomens partially exposed were selected and placed in 96-well racks, and the CBBs boring into the fruits were treated directly. After 20 days, the fruits were dissected, and the CBB biological stages inside each fruit were recorded. The GFs served as substrates that mimic natural conditions to evaluate toxic, chemical, and biological insecticides against the CBB.

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

A method using green coffee fruits (GFs) was developed to test the toxicity of insecticides against the coffee berry borer (CBB). Insecticides or toxic substances were applied to disinfected GFs before or after CBB infestation. Insect mortality, repellency, and reproductive capacity, in addition to other parameters, were evaluated.

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Biology

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild Monochamus alternatus Populations for Biocontrol Applications in Forest Wood Borers

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

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

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Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Building Double-decker Traps for Early Detection of Emerald Ash Borer

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

A Method for Quantifying Foliage-Dwelling Arthropods

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Bioassays for Monitoring Insecticide Resistance

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer