O protocolo de Havaí para acompanhamento científico da broca do café: um modelo para agroecossistemas de café no mundo

Coffee is an economically important crop for Hawaii, and Hawaii coffee, especially from Kona, is world-renowned. More recently, coffee produced in the Ka'u district on the island of Hawaii, the Big Island, has been recognized for its exceptional quality. Part of the reason for this high quality is that coffee grown in Hawaii has historically been isolated from many of the pests and diseases that affect other coffee-growing regions of the world.

However, there is a new threat to this important crop in the islands. Coffee berry borer, also called CBB and the most serious pest of coffee worldwide, was detected on the Big Island of Hawaii in 2010. It has now established on the Big Island, Maui, and Oahu, and if control measures are not used CBB can completely ruin the crop as it feeds on the coffee bean.

In Hawaii, the most important control measure against coffee berry borer, or CBB, is good sanitation. Sprays of the biopesticide Beauveria bassiana, a fungus that kills insects, are also widely used. One of the difficulties is that this tiny beetle spends most of its life within the seed of a coffee berry, protected from pesticide sprays.

Monitoring the distribution and abundance of CBB within a coffee field is key to its efficient control. We developed this scientific protocol to collect data on movement, infestation rates, mortality factors, and plant phenology. We are doing this with the aid of a mobile, electronic data recording application.

The goal of the complete protocol is to provide robust data for scientific research on CBB control. However, elements of this approach are appropriate for management-oriented monitoring as well. Generate a map of the coffee field, and divide the field into zones, each about 335 meters square.

These will be used to ensure a systematic random sampling design across the field. Using an electronic data collection platform, such as Fulcrum, build a data collection application comprised of the following interlinked databases:traps, zones, site service, weather stations, and management. These databases will be used in all subsequent steps of the protocol for the collection and organization of data.

Determine the number of traps needed to monitor CBB movement in each field. Trap density per field should approximate five traps for small fields and 10 traps for large fields. Using a thumbtack, make a series of drainage holes above the fill line in each trap collection cup to avoid dilution of the kill solution by rainwater.

Assemble the funnel traps according to the manufacturer's instructions. Prepare a liter of kill solution comprised of 200 milliliters of propylene glycol and 800 milliliters of water. Next, prepare an attractant mixture comprised of a three-to-one solution of methanol to ethanol.

Pour 40 milliliters of the attractant into plastic semi-permeable bags, and place in a container for transport. Deploy the traps by randomly distributing them across the field. Traps should be placed 5 to 1.5 meters above the ground and clear of the aisles.

Stakes may be used to effectively secure traps between trees. Write the site name and trap number with permanent marker on each trap for future identification. Fill trap collection cups with 100 milliliters of the glycol kill solution, and screw the cups tightly into place.

Attach a paper clip to each attractant bag, and use the paper clip to hook the bag to the center of the trap. Using a mobile device equipped with the electronic data collection platform, navigate to the traps database and create a new deployment record comprised of the site, date, trap number, and a photograph of the trap. The location of the trap within each field is automatically recorded via GPS on the mobile device.

Upon arrival in the field, navigate to the site service database within the electronic system, and create a new site service record comprised of the site name, date, and field technician name. Locate the trap in the field. Place a fine-mesh hand sieve onto a plastic container, and pour the kill solution from the collection cup through the sieve.

Transfer the kill solution back to the collection cup, and swish the liquid around to ensure that all the CBB are removed from the collection cup. Within the new site service record, navigate to the trap service database and create a new trap service record. Enter the relevant trap number, and photograph the sieve with the site name and trap number in the background.

Save the photograph to the trap service record. Using a spoon or metal spatula, scoop all insects into a vial filled with 70%ethanol. Label the vial with site, date, and trap number.

Refill the collection cup with fresh kill solution and screw back onto the trap. Within the site service record, navigate to the zone service database and create a new zone service record. Select a sampling zone from the site map in the linked zones database.

To avoid sampling bias, randomly select a tree from within the zone by casting eyes downward so only the bases of the trees are visible. Standing in front of the selected tree, randomly choose a lateral branch around chest height. Clip a ruler onto the selected branch, making sure that the ruler does not block any of the reproductive parts from the camera's field of view.

Take a single photo ensuring that the ruler and the entirety of the target branch are visible. Take a second photo of the whole tree. Try to get as much of the mid-level canopy in the photo as possible.

Save both phenology photos to the zone service record. If the branch used for phenology appears to have greater than 30 green berries, count the number of berries on the branch that are at least pea size and larger and are green to light yellowish-green in color. Enter this number into the zone service record.

Also in the zone service record, enter the number of green berries infested by CBB on the branch. Infested berries will have a small hole that is typically located in the central disc. CBB may or may not be visible in the hole.

Enter the number of green infested berries with visible white Beauveria bassiana fungus. The fungus may be seen on CBB and/or surrounding the entrance hole. Enter the number of raisins on the branch.

Collect three infested green berries from the branch. Place infested green berries in a plastic container, and label with site and date. Containers should be stored in a cooler on ice until they can be transported back to the lab.

Place a coarse-mesh hand sieve over a plastic container, and empty the beetles from the collection vial into the sieve. Use a wash bottle filled with water to get all of the contents out of the vial. Use the wash bottle to spray the contents in the sieve, forcing as many small insects through the sieve as possible.

This permits larger insects and debris to be separated out from the small beetles in the sample and limits inaccuracies in volumetric estimates of CBB. Discard the large insects and debris, and rinse out the sieve. Place a fine-mesh hand sieve over the second plastic container, and pour the contents of the first container into the fine-mesh hand sieve.

If there are more than several hundred CBB, skip ahead to step 7.6. If there are less than several hundred CBB, place the fine-mesh sieve onto a paper towel to remove excess water. Turn the sieve upside down, and tap all the contents onto a clear plastic lid.

Spread the beetles around with a paintbrush if they are clumped together, and allow them to sit until dry. If there are less than several hundred CBB, use a fine-tipped paintbrush or similar implement to line the beetles up in rows that are several beetles wide, and begin counting under a light microscope. Count the total number of beetles, and separate into CBB and other categories.

If there are more than several hundred CBB, transfer the CBB from the fine-mesh sieve to a 10-mil syringe using a metal spatula. Place the ejector column into the syringe, and press down gently until you feel slight resistance, being careful not to crush the beetles. Record the volumetric value on the syringe.

Count 200 beetles from the volumetric sample using the protocol described above. Use the following equations to determine the number of CBB versus other beetles in the sample. When the trap count has been completed, navigate to the relevant trap service record and enter the number of CBB and other beetles.

Export the coffee phenology photographs from the data collection application. Open the photograph, and locate the branch with the attached ruler. For this branch score the following:number of nodes, presence or absence of immature buds, mature buds, candles, open flowers, and pinheads, number of pea-sized green berries, immature green berries, mature green berries, berries showing a color break, fully ripe berries, and raisins.

Take the infested green berries out of cold storage, and allow them to warm to room temperature for 10 to 15 minutes before proceeding with berry dissection. Dissection of the infested berries allows the position of the adult CBB to be determined. Position AB indicates the female has initiated penetration into the berry but has not reached the endosperm.

Position CD indicates the female has entered the endosperm. Using a scalpel or similar implement, make a slice through the berry parallel to the central disc as a preliminary assessment of beetle position. Next, make a series of shallow slices perpendicular to the central disc and around the entrance hole to determine if the CBB is in the AB or CD position.

Subdivide the AB and CD categories into alive, dead by Beauveria bassiana, dead by other causes, and beetle missing. If it is unclear if the adults are alive or dead, zoom in with a microscope and watch the legs for movement. Place counted individuals into a dish with water or alcohol.

This helps to keep track of what has been counted and prevents adult beetles from escaping into the lab. Once the dissections for a site are completed, navigate to the berry dissection database in the relevant site service record and enter the total number of CBB in each category. Dissected samples should be placed in a container and frozen for 72 hours before disposal.

Servicing traps on a weekly versus biweekly basis revealed that biweekly sampling is sufficient for capturing general trends in CBB flight activity throughout the growing season on Hawaii Island. However, if high-resolution data on CBB movement is desired, a weekly trap servicing may be done. For a sample farm located in Kona, the mean number of CBB caught per trap per day revealed a total of four CBB flights throughout the growing season, with major peaks in activity occurring in March and December.

Data collected during a monitoring survey in June at a sample farm in Kona revealed a high concentration of CBB in the central portion of the farm. Here, the size of the red circle is proportional to the number of green infested berries on a sampled branch. A total of 25 branches were sampled during the survey, and a range of zero to 36 infested green berries were observed per branch.

The majority of infested berries dissected early in the growing season hosted CBB in the AB position, while the majority of berries dissected later in the season hosted CBB in the CD position. Following a peak in berry production, harvesting of ripe coffee cherries occurred from late July to December. Finally, seven applications of Beauveria bassiana were done at approximately one-month intervals throughout the season, with CBB mortality observed to range from zero to 23%All the data collected is backed by a network of weather stations and management records.

Together, this methodology and associated tools will facilitate better control methods for CBB, both here in Hawaii and wherever this monitoring method is used around the world.