Objectives: To determine the use patterns and relative rank of host plant species used by Homalodisca coagulata and other xylophages for adult feeding. Determine frequency of leafhoppers/plant in the field. Determine the diurnal pattern and rate of leafhopper feeding (excretion).

Methods: Collect or rear leafhoppers from the test hosts or from crape myrtle or other herbaceous plants. Field collected insects may be infected with Xylella fastidiosa (XLB). Collecting nymphs in late instars in the field and rearing to the adult stage on uninfected plants will insure that they do not carry the XLB. Host plants such as soybean or black-eyed peas can be use for nymph rearing.

Use the feeding assemblies (Figure 1) to cage leafhoppers on the plants of interest and to collect and measure their excreta. Leafhoppers should be observed in the field or caged (cloth sleeves) on the plants for few days prior to the bioassay if they are collected from host species other than the one to be tested. The cages should enclose the current and part of the previous year's growth. Determine the proper placement for the assembly by observing where on the branch the leafhoppers are feeding naturally.

Figure 1. Leafhopper feeding assembly parts. (see footnote)

Using an aspirator (8-inch piece of 8 mm diameter glass tubing with cloth over one end attached to a 4-foot piece of tubing), place one or more female leafhoppers (males feed >25% less than females) in each assembly and cover it with aluminum foil to block the sun, rain and dew. Make sure the aluminum foil is wrapped tightly at the top (bottom can be open to admit light) to deflect water that may run down the limb into the assembly. Assemblies should be placed on the plants the previous day and emptied, or in the morning to prepare for the maximum feeding period of midday to early afternoon (Figure 2). Use five to 10 assemblies per species/cultivar tested, preferably on several different plants. Five plants with two assemblies per host plant is adequate.

Figure 2. Leafhopper feeding assemblies on crape myrtle.

Bioassays should be conducted during the time of year when leafhoppers are most frequently seen feeding on the host plant species in the experimental area . For example, this is approximately the first two weeks in June for peach in north Florida.

Leafhoppers placed in the assemblies should begin feeding immediately. If the leafhoppers do not stay in the assemblies, but instead move down into the 15 ml collection vial, it is an indication that they do not like the host plant (primary) or the site on the branch (secondary) on which they were placed (see above). Handling leafhoppers often is detrimental to them and some of the leafhoppers may die doing the experiment. If you cage (condition) them on the same host plant species you are testing, you should not have a problem with the leafhopper behavior in response to a new host. Thus, you can replace dead leafhoppers with ones from the stock cage as needed and still collect reliable data.

Run the bioassay for three to five days to establish the feeding (excretory) pattern. Higher amounts are fed/excreted on quality host plants. Record and empty the excreta at least three times/day: 10 AM, 12 PM and 2 PM. More defined data will result from including 8 AM and 6 to 8 PM data points. On most host plants leafhoppers feed during the day, however, on certain plant species they will feed at night in response to plant nutrient cycles. If night feeding is dominant, run the bioassay accordingly.

At the same time the assemblies are used to determine feeding, counts of adult and nymph leafhoppers should be made on five to 10 host plants of the species in the field. The daily flight activity pattern observed in Florida for H. coagulata is also included (Figure 4). This pattern can be developed using yellow sticky traps placed about 1 to 2 meters off the ground.

Note of Caution: The relationship of vector plant preference to XLB susceptibility or preference has not been established. On grape in California, Purcell showed that leafhopper abundance was not related to XLB infectivity rate. In fact the relationship may be such that reduction in the abundance of feeding leafhoppers by using germplasm that is not preferred by the vector is relatively unimportant. This remains to be elucidated. We have related leafhopper abundance to feeding rate (R²=0.63, P=0.0001) for crape myrtle, peach and plum during peak leafhopper appearance on these hosts. We have also documented that this bioassay supports the observations and results based on XLB sampling of plum cultivars by Dr. J. Norton of Auburn University in Alabama. The feeding rate of the leafhopper H. coagulata using the feeding bioassay, was highly correlated with Norton's assessment of XLB susceptibility and resistance. We have documented a strong relationship of leafhopper feeding rate to plant xylem nutrient content that also may be very important to the XLB. We have also documented a strong relationship of nymph survival to host plant chemical profiles (different from adult needs) that may provide another management tool.

Figure 1: Feeding assembly instructions. The assembly is placed over a small branch of the host plant and covered carefully with aluminum foil to block sunlight and water from entering the assembly. The ventral surface is not usually covered. The assembly is made with a 50 ml polypropylene thin wall centrifuge tube (Nalgene 3150-0050), a 15 ml graduated tube with snap cap (Falcon 2057) and a 20 to 25 mm tissue culture sponge stopper. A 5 mm hole is cut at the end of the tube on opposite sides just above the bottom curvature. The centrifuge tube is split from the open end to the hole on one side. The center of the 15 ml tube cap is removed such that the portion overlapping the vial when inserted remains intact to prevent leakage. The cap is then glued over the hole opposite the split on the centrifuge tube. The vial is attached to make up the excreta- collecting portion of the assembly.

The assembly can then be placed over a small branch by placing the branch through the split and into the hole. The sponge is split to the center on one side so that it can be placed over the branch and then into the centrifuge tubes open end to stopper the tube and hold the leafhopper. Leafhoppers can be placed in the sponge end or through the hole into the vial while its removed. The whole assembly is then covered with aluminum foil as shown with the leafhopper inside. Xylophages produce copious quantities of excreta that is mostly water that is collected in the 15 ml tube. With this design feeding (excretion) rates can be determined in the field or anywhere else under any most weather conditions.