This method uses green coffee fruits to test the toxicity of insecticides against the coffee berry borer. The insecticide application is performed before or after the insect infestation to study the insect mortality repellency and other effects. Using real green coffee with nutrients adequate for insect growth is the most appropriate way to evaluate the toxicity of compounds to insects under simulated natural conditions.
The protocol allows the evaluation of the toxic effects of chemical insecticides and toward pathogens such as fungi or repellent substances. This infection of the fruits and the insects is the critical step as improper disinfection can cause insect death due to contamination. Insect quality is also important as weak insects will overestimate the results of a toxic product.
Visual demonstration allows identification of the appropriate stage of the fruits the insect infestation process, the appropriate moment to spread the toxic substance. And the way for dissecting the fruits. Demonstrating the procedure will be Claudia Martinez, a research assistant from the laboratory.
To begin, collect the green coffee fruits early in the morning with the approximate developmental age of 120 to 150 days after flowering from coffee plantations. Select around 300 uniformly-sized and healthy fruits and withdraw the peduncles. Dip the green coffee fruits into a 2%soap solution and clean thoroughly by rubbing them.
Then rinse the fruits three times with clean water. Immerse them in 0.5%sodium hypochlorite solution and place them on the shaker for 10 minutes at 110 revolutions per minute. Rinse the fruits with clean water three times and dry them with sterile paper towels.
Irradiate the fruits by placing them at 55 centimeters from the UV source for 15 minutes, inside a UV-enabled horizontal laminar flow station. Move the fruits after every five minutes to ensure the proper irradiation of the whole fruit. To set up the bioassays, immerse the coffee berry borers that emerged on the same day in 0.5%sodium hypochlorite solution and agitate them slowly with a brush for 10 minutes.
Filter these insects through a muslin cloth and wash them three times with sterile distilled water. Remove excess water with sterile paper towels. Prepare a group of 30 green coffee fruits per experimental unit and place them in a plastic box.
Apply different concentrations of a test product for evaluation using a portable sprayer unit, here, 5%and 6%alkaloid emulsions were used. As a control, spray a group of green coffee fruits with water. Utilize at least three experimental units per treatment, spraying one after another.
In a sterile hood, release two coffee berry borer adults per green coffee fruit, and cover the boxes after 30 minutes. Leave the plastic boxes with the infested green coffee fruits in the dark inside a room or incubator, having controlled conditions. After 1, 7, 15, and 21 days, count the number of borer fruits and living and dead insects outside the fruits in each box.
At 20 days post-infestation, dissect each green coffee fruit under a stereo microscope with 10x magnification and count the number of healthy seeds or seeds damaged by the insects in each fruit. Count the different coffee berry borer biological stages. Observe the number of dead insects in each seed to determine insect mortality per experimental unit.
In a sterile hood, release coffee berry borer adults to the previously disinfected green coffee fruits in a two-to-one ratio and allow the infestation to proceed for three hours at 21 degrees Celsius. Most green coffee fruits should be infested after three hours with the abdomen of the coffee berry borer still exposed. Select 46 infested fruits and place them in a 96-well plastic rack so that the treatment can be directly sprayed on the coffee berry borer perforating the fruit.
Spray three racks at least three times for treatment, one after the other and cover the racks after 30 minutes. After 20 days, dissect the green coffee fruits under a stereo microscope at 10 x magnification and count the number of healthy seeds or seeds damaged by the insects in each fruit. Count the different coffee berry borer biological stages and the number of dead insects in each seed to determine insect mortality per experimental unit.
Follow the initial steps for evaluating a product with a protective effect on the fruits and release the green coffee fruits adults into the plastic boxes. Then count the number of coffee berry borers that fly away from the boxes and the number that infest the green coffee fruits. After the incubation, under controlled conditions, followed by dissection at 20 days post-infestation and counting the seeds and dead insects, follow the steps outlined for evaluating the product, after the coffee berry borer infestation.
Count the number of coffee berry bores that moved out of the green coffee fruits, or flew away from the fruits. After spraying each treatment, then follow the steps from incubation under controlled conditions to determine the insect mortality per experimental unit. 5%and 6%insecticide applied to the coffee berry borer, caused high insect mortality on day 20, and showed significant differences compared to control.
It was seen that the application of insecticide two resulted in 94%of healthy seeds, and insecticide one resulted in 89%of healthy seeds. 37%of the seeds were not infested in the control group. In the case of post-infestation, the products penetrated the insect cuticle causing insect mortality.
The highest mortality occurred with insecticide two. The effects of the insecticides were reflected in the percentage of healthy uninfected seeds, on day 20 of the evaluation. A reduced insect population was found inside the coffee seeds compared with the insect population found in the control group, sprayed with water.
Entomopathogen and repellent combined action resulted in minimum seed damage and maximum insect mortality. This infection of the fruits and disinfection of the insects ensure the protocol success. This methodology also can be used for evaluating the tolerance or resistance of coffee fruits to the coffee berry borer.
A negative effect can be also observed against the insect population in the case of insect-resistant coffee seeds. This methodology can be used not only in entomology for evaluating insecticide with different modes of action but also in plant breeding for evaluation of tolerant varieties and plant pathology to study the fruit/pathogen interaction.
