Name: Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example (Video) | JoVE Journal
Uploaded: 2011-06-19T14:00:00.000+00:00
Duration: 6 min 52 s
Description: Learn to characterize herbivore resistance mechanisms using a no-choice test with spittlebugs on Brachiaria grasses. This method estimates antibiosis and tolerance contributions for breeding durable pest resistance.

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5 minutes

This production and the experimental work presented herein reflect the devoted and enthusiastic help of Reynaldo Pareja kindly supported by Lina Aguirre, Gilberto C&oacute;rdoba, William Mera, Ximena Bonilla, and Dar&iacute;o Viveros. We also thank John Miles and three anonymous reviewers for comments that helped us improve the video and manuscript.

1. Plants
<ol>
	<li>Single stem cuttings from mature plants are used as experimental units. The cuttings are trimmed to 10 cm to ensure uniformity of planting material. </li>
	<li>To prevent contamination by plant pathogens, the cuttings are washed in a 3 % sodium hypochlorite solution for 2 min and then rinsed thoughroughly with tap water.</li>
	<li>Each cutting is planted in ca. 36 g of sterile soil in a cylindrical PVC tube (5.3 cm outside diameter by 6.5 cm height) sealed at the lower end with a Styrofoam cup and capped with a PVC bushing. The cutting is held in place by a foam ring placed in the central opening of the bushing.</li>
	<li>Plants are fertilized and watered as needed to maintain adequate soil moisture. </li>
	<li>Plants will grow for one month before being challenged by insects, allowing development of superficial roots, which serve as feeding sites for the root-xylem-feeding spittlebug nymphs.</li>
	<li>PVC tubes with rooted cuttings are inverted for 8 d prior to infestation to stimulate further the growth of superficial roots, partially adjusting for inherent differences in root architecture. This step requires an artificial source of light, which we provide for 24 h a day. To avoid confounding escapes from herbivory with herbivore resistance, individual plant replicates with few or no superficial roots after this inversion treatment are eliminated from the experiment. This elimination should be infrequent, never leading to the elimination of the genotype they represent. </li>
</ol>
2. Insects
<ol>
	<li>Adult spittlebugs are collected from the field, identified to species, and introduced to oviposition cages where they are provided foliage of susceptible brachiariagrasses to feed on. Females lay their eggs in soil contained in a shallow removable tray placed on the bottom of the cages. </li>
	<li>After several days, the soil is suspended in water and passed through a series of sieves (42, 60 and 150 mesh) to collect the eggs. Suspending the sieved material in a 30% saline solution causes the mature eggs to float, leaving behind immature eggs and any residual organic matter. </li>
	<li>To prevent contamination by insect pathogens, eggs are disinfected in a 0.5 % sodium hypochlorite solution for 5 min and then rinsed with distilled water. </li>
	<li>Each test plant (experimental unit) is infested at the soil surface with six mature eggs. Only mature eggs close to hatching are selected for infestations. They can be identified by the presence of two red dots in their anterior part, corresponding to the insect’s eyes; pink marks in their posterior part, corresponding to the insect’s abdomen; and a fully-expanded operculum. </li>
	<li>Successful hatching is confirmed 4 d later, at which point any unhatched egg is replaced by a neonate spittlebug from our colony. </li>
	<li>Test plants are arranged in a randomized complete block design with six replications per genotype.</li>
</ol>
3. Evaluations
<ol>
	<li>The experiment is evaluated ca. 30 d after infestation (exact times vary depending on the insect species studied).</li>
	<li>Damage is scored on a 1 to 5 visual scale, where 1 corresponds to no visible damage and 5 corresponds to a dead plant (Figure 1). Scoring plant damage provides an estimate of tolerance.</li>
</ol>
<img src="/files/ftp_upload/3047/3047fig1.jpg" alt="Plant growth experiment, five stages, numbered plants, biological research, growth comparison." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Damage scale used to assess tolerance to spittlebug herbivory in Brachiaria spp. 1 = no detectable damage; 2 = 0-25% necrotic foliage; 3 = 25-50% necrotic foliage; 4 = 50-75% necrotic foliage; 5 = 75-100% necrotic foliage. 
<ol start="3">
	<li>Insect survival is assessed by counting the insects reaching either their final instar or adult stage (Figure 2). Scoring for insect survival provides an estimate of antibiosis.</li>
</ol>
<img src="/files/ftp_upload/3047/3047fig2.jpg" alt="Insect life cycle stages diagram with developmental size progression." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Spittlebug development from first-instar (left) to adult (right). Only insects reaching at least their final instar (arrows) at the date of evaluation are counted as "survivors." 

4. Representative results
Results from a representative assay screening Brachiaria hybrids for resistance to the spittlebug Aeneolamia reducta are presented in Figure 3. 
<img src="/files/ftp_upload/3047/3047fig3.jpg" alt="Scatter plot; plant damage vs. insect survival; ecological study; data pattern analysis." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Results from screening Brachiaria hybrids for resistance to spittlebug Aeneolamia reducta. We consider plants resistant if their response to herbivory falls in the bottom half of this plot. Antibiosis increases from right to left and tolerance increases from top to bottom. The arrows point at our resistant checks.

<ol><li>Painter, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPainter%2C+R+%22Insect+resistance+in+crop+plants%22">Insect resistance in crop plants</a>. , Mcmillan. (1951).</li>
<li>Kogan, M., Ortman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antixenosis-a+new+term+proposed+to+define+Painter%27s+%27Nonpreference%27+modality+of+resistance%5BTitle%5D)+AND+%22Bull.+Entomol.+Soc.+Am%22%5BJournal%5D)">Antixenosis-a new term proposed to define Painter's 'Nonpreference' modality of resistance.</a> Bull. Entomol. Soc. Am. 24, 175-176 (1978).</li>
<li>Kennedy, G., Gould, F., Deponti, O., Stinner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ecological%2C+agricultural%2C+genetic%2C+and+commercial+considerations+in+the+deployment+of+insect-resistant+germplasm%5BTitle%5D)+AND+%22Environ.+Entomol%22%5BJournal%5D)">Ecological, agricultural, genetic, and commercial considerations in the deployment of insect-resistant germplasm.</a> Environ. Entomol. 16, 327-338 (1987).</li>
<li>Holmann, F., Peck, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Economic+damage+of+grassland+spittlebugs+%28Homoptera+Cercopidae%29+in+Colombia%3A+a+first+approximation+of+impact+on+animal+production+in+Brachiaria+decumbens%5BTitle%5D)+AND+%22Neotrop.+Entomol%22%5BJournal%5D)">Economic damage of grassland spittlebugs (Homoptera Cercopidae) in Colombia: a first approximation of impact on animal production in Brachiaria decumbens.</a> Neotrop. Entomol. 31, 275-284 (2002).</li>
<li>Miles, J. W., Cardona, C., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recurrent+selection+in+a+synthetic+brachiariagrass+population+improves+resistance+to+three+spittlebug+species%5BTitle%5D)+AND+%22Crop+Sci%22%5BJournal%5D)">Recurrent selection in a synthetic brachiariagrass population improves resistance to three spittlebug species.</a> Crop Sci. 46, 1088-1093 (2006).</li>
<li>Ferrufino, A., Lapointe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host+plant+resistance+in+Brachiaria+grasses+to+the+spittlebug+Zulia+colombiana%5BTitle%5D)+AND+%22Entomol.+Exp.+Appl%22%5BJournal%5D)">Host plant resistance in Brachiaria grasses to the spittlebug Zulia colombiana.</a> Entomol. Exp. Appl. 51, 155-162 (1989).</li>
<li>Lapointe, S. L., Serrano, M. S., Arango, G. L., Sotelo, G., Cordoba, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibiosis+to+spittlebugs+%28Homoptera%3A+Cercopidae%29+in+accessions+of+Brachiaria+spp%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Antibiosis to spittlebugs (Homoptera: Cercopidae) in accessions of Brachiaria spp.</a> J. Econ. Entomol. 82, 1764-1766 (1992).</li>
<li>Cardona, C., Miles, J. W., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+methodology+for+massive+screening+of+Brachiaria+spp.+genotypes+for+resistance+to+Aeneolamia+varia+%28Homoptera%3A+Cercopidae%29%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">An improved methodology for massive screening of Brachiaria spp. genotypes for resistance to Aeneolamia varia (Homoptera: Cercopidae).</a> J. Econ. Entomol. 92, 490-496 (1999).</li>
<li>Cardona, C., Fory, P., Sotelo, G., Pabon, A., Diaz, G., Miles, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibiosis+and+tolerance+to+five+species+of+spittlebug+%28Homoptera%3A+Cercopidae%29+in+Brachiaria+spp.%3A+implications+for+breeding+for+resistance%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Antibiosis and tolerance to five species of spittlebug (Homoptera: Cercopidae) in Brachiaria spp.: implications for breeding for resistance.</a> J. Econ. Entomol. 97, 635-645 (2004).</li>
<li>Smith, C., Khan, Z., Pathak, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSmith%2C+C+%22Techniques+for+evaluating+insect+resistance+in+crop+plants%22">Techniques for evaluating insect resistance in crop plants</a>. , CRC Press. (1994).</li>
<li>Lapointe, S., Sotelo, G., Arango, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+technique+for+rearing+spittlebugs+%28Homoptera%3A+Cercopidae%29%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Improved technique for rearing spittlebugs (Homoptera: Cercopidae).</a> J. Econ. Entomol. 82, 1764-1766 (1989).</li>
<li>Lopez, F., Cardona, C., Miles, J. W., Sotelo, G., Montoya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Screening+for+resistance+to+adult+spittlebugs+%28Hemiptera%3A+Cercopidae%29+in+Brachiaria+spp.%3A+methods+and+categories+of+resistance%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Screening for resistance to adult spittlebugs (Hemiptera: Cercopidae) in Brachiaria spp.: methods and categories of resistance.</a> J. Econ. Entomol. 102, 1309-1316 (2009).</li>
</ol>

No conflicts of interest declared.

Discriminating among herbivore resistance mechanisms can illuminate crop breeding efforts3. Resistance based on strong antibiosis could, under some circumstances, create selection pressures for more aggressive pest biotypes. On the other hand, resistance based on tolerance could allow pest populations to increase until tolerance eventually is overwhelmed. Breeding for durable resistance, therefore, requires careful consideration of what resistance mechanisms would confer most stability for a particular crop/pest system [see Kennedy et al. (1987) for a comprehensive discussion on the topic]3.
This video demonstration builds upon several previous studies designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in brachiariagrasses6-9. In contrast with choice tests or field experiments, no-choice tests ensure all plants receive the same herbivore pressure, thus providing control for differences arising from herbivore behavior (e.g., host preference, aggregation). For this reason, however, no-choice tests are unsuitable to assess resistance by antixenosis. 
Artificially rearing insect colonies is the preferred method to obtain test insects for resistance screening assays10. Colonies provide a dependable and uniform source of spittlebugs of a known age for experiments to be conducted any time of the year11. It may, however, be necessary to periodically infuse wild individuals into the colony to ensure that the colony does not deviate genetically from relevant field populations10. To assess the fitness of these artificially-reared herbivores, and monitor possible changes in their aggressiveness over time, it is important that all screenings include suitable checks of known resistance levels. 
Multiple parameters must be carefully defined to design an appropriate resistance screening assay. A few key parameters we have considered to design our test include the age of the host plants, the species and developmental stage of the spittlebugs, the infestation level, and the duration of plant-insect contact8,9,12. Several rounds of experimentation and protocol refinement may be needed to reach to a rapid, cost-effective, reliable, assay that adequately predicts herbivore resistance in the field.

characterizing herbivore resistance mechanisms: spittlebugs on brachiaria spp. as an example

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.

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 ...

Hi, my name is Ush Bara and I'm an entomologist at the International Center for Tropical Agriculture. The overall objective is video demonstrate a method to characterize mechanisms of host plant resistance to herbo in support of cooperating efforts, our breeding initiative to enhance spit bug resistance. In aria, grasses will provide an example.Spit bugs are serious pests of many economically important grasses, including tropical pastures, sugar cane, and corn. In the American tropics pastures belonging to the gen bacteria are their most important host. A plant can resist herbivore damage through three broad mechanisms.Osis, antibiotics, intolerance, antis, sometimes called non preference is a degree to which the plant is avoided when the hoverboard is able to select plants. Anti osis is typically detected in choice cyst where heavy boards are given access to more than one genotype. Antibiotics is a degree to which the plant affects the fitness of the herbo feeding on it.Finally, tolerance is the degree to which the plant can withstand or repair damage caused by the HBO without compromising the HBO's growth and reproduction. Both antibiotics and tolerance are best detected in no choice tests where hbos are confined and therefore forced to feed on a single plant. Our video will demonstrate a no choice test designed to estimate antibiotics intolerance, spit bug IV.In aria grasses host plan resistance is a key spit bug management tactic.However, resistance based on strong antibiotics could create selection pressures for more virulent pest biotypes. On the other hand, resistance based on tolerance would allow spit dog populations to continue growing until the numbers overwhelm the plant. Durable resistance therefore may require some levels of both antibiotics and of tolerance.This protocol will demonstrate how to estimate the relative contributions of each to spit bug resistance. In Aria Aria genotypes are a plane from the germ Plasm Bank of the International Center for Tropical Agriculture. The plants are vegetatively propagated from single stem cuttings, from mature plants used as experimental units.The cuttings are trimmed to 10 centimeters to ensure uniformity of planting material to prevent contamination by plant pathogens. The cuttings are washed in a 3%sodium HypoChlor solution for two minutes and then rinse thoroughly with tap water. Each cutting is planted in approximately 36 grams of sterile soil in A PVC tube sealed with a lower section of a styrofoam cup and capped with a PVC bushing.The cutting is held in place by a foam ring placed in the central opening of the bushing. Plants are fertilized and water is needed to maintain adequate soil moisture. The plants will grow for one month before being challenged by insects, allowing development of superficial roots, which serve as feeding sites for root feeding.Spittle bog NIMS inverting the plants for eight days prior to infestation, further stimulates the growth of superficial roots, partially adjusting for inherent differences in root architecture. This step requires an artificial source of light, which we provide for 24 hours a day. Plans with little or no superficial roots.After this inversion treatment are eliminated from the experiment. 30 days after planting, each plant is infested at the soil surface with six mature eggs obtained from our spittle bug colonies To establish these colonies, adult spittle bugs are collected from the field identified to species, and introduced to a position cages where they're provided foliage of susceptible aria to feed on. Females lay their eggs in soil contained by a shallow removal trail placed on the bottom of the cages.After several days, the soil is suspended in water and passed through a series of sieves to collect the eggs. Suspending deceived material in a 30%saline solution causes the mature eggs to float, leaving behind immature eggs on any residual organic matter. To prevent contamination by insect pathogens ex are disinfected in a 0.5%sodium hypochlorite solution for five minutes and then rinse with distilled water only mature eggs closely hatching are selected for infestations.They can be identified by the presence of two red dots in the anterior part corresponding to the insects eyes. Pink marks in the posterior part corresponding to the insects abdomen, and a fully expanded a perm. Successful hatching is confirmed four days later, at which point any unhatched egg is replaced by a neonate spit block.From our colony test. Plants are arranged in a randomized complete block design with six replications. The experiment is evaluated approximately 30 days after infestation.Scoring for plant damage provides an estimate of tolerance. While scoring for insect survival provides an estimate of antibiotics. Damage is scored using a one to five visual scale where one corresponds to no visible damage and five corresponds to plant death.In turn, survival is assessed by counting the insects reaching either their final ins insert or their adult sage. To categorize resistance mechanisms, it is useful to visualize results using a by plot where insect survival is on the x axis and plant damage on the Y axis. Antibias increases as one moves from right to left.On the other hand, tolerance increases as one moves from top to bottom. We consider plants resistant if the response to herbivore falls in the bottom half of this plot. If this response falls on the left quadrant, the dominant resistance mechanism is antibiotics.If the response falls on the right quadrant, the dominant resistance mechanism is tolerance. Here's a representative sample of our screening data. The points on the top represents susceptible plans.The points on the lower left quadrant represent plans resistant by antibiotics. The points on the lower right quadrant represent plants resistant by tolerance. We've just watched a demonstration of a no choice assay to estimate the relative contributions of antibiotics, intolerance to plant resistance to however works.We use spit bug resistance in Aria as an example, but the principles presented should be relevant to other systems. We hope our video helps you in your own efforts. Good luck.

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Learn to characterize herbivore resistance mechanisms using a no-choice test with spittlebugs on Brachiaria grasses. This method estimates antibiosis and tolerance contributions for breeding durable pest resistance.

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

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