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

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.