α-Copaene is a powerful repellent against the insect that transmits citrus greening

The compound is 100 times more effective than β-caryophyllene against the disease impacting orange groves in Florida and threatening citrus growers in the state of São Paulo, Brazil, the world’s largest orange producer.

By Karina Ninni  |  Agência FAPESP – Greening is the most devastating disease in citrus farming. In São Paulo, the Brazilian state that produces the most oranges in the world, 44% of orange trees have the disease. Since its discovery in Brazil in 2004, efforts to combat greening have focused on controlling the citrus psyllid (Diaphorina citri), which spreads the disease, managing insecticides, planting seedlings that are free of the Candidatus Liberibacter asiaticus bacteria that cause it, and removing diseased trees from orchards.

In an attempt to manipulate the insect’s behavior, a team of scientists discovered α-copaene, a molecule found in large quantities in copaiba oil. They found that it is highly effective at repelling psyllids – in fact, it is 100 times more potent than the previously investigated substance, β-caryophyllene.

“There’s no effective way to kill the bacteria in the plant. If the plant is infected, it must be eliminated and removed from the orchard because it becomes a source of contamination for the psyllid that transmits the disease. However, we’ve identified another insect repellent compound, in addition to the one we’d already identified,” explains Rodrigo Facchini Magnani, a chemist from the Research and Development Department of the Citrus Defense Fund (FUNDECITRUS) and one of the authors of a recent paper on the subject published in Scientific Reports.

It takes about six months for the orange tree to show symptoms of the disease. The PCR technique is used to detect the bacteria in the plant and confirm the disease diagnosis. The most economical way to determine contamination is through visual inspection. There are several symptoms: the leaves develop yellow spots (mosquito spots), the fruit becomes asymmetrical, the seeds abort, and the juice produced is inferior. Over time, the bacteria multiply in the plant, contaminating the branches. Year after year, this increases fruit drop and reduces orchard productivity. The plant becomes unproductive, and worse, it serves as a source of inoculation for new generations of psyllids.

Magnani says that, in 2009, a national commission was formed with representatives from academia, local producers, and industry to investigate the disease, which had already spread to the state of São Paulo. The commission was in constant contact with an international counterpart that had been monitoring the disease worldwide. “At some point, small-scale producers in Vietnam observed and reported that when guava trees were interspersed with tangerine plants, there was a smaller insect population and a lower incidence of the disease in the tangerine plants. This caught the attention of the scientific community, industry, and producers,” the researcher recalls.

The guava tree may emit compounds that interfere with the insect’s approach to the plants. After investigation, a compound was found in large quantities in the aroma of guava trees: β-caryophyllene. As the research progressed, it was concluded that it did indeed repel the insect and that this could be used as a strategy in citrus plants themselves, causing them to increase their production of this compound (since citrus fruits already produce the compound, but in smaller quantities).

“Initially, we inserted caryophyllene-producing genes into Arabidopsis plants, which grow quickly. Arabidopsis is a model widely used in molecular biology and genetic engineering and also naturally produces a very small amount of β-caryophyllene. After the experiment, the plant not only overexpressed the compound but also repelled the insect. However, when this gene is introduced, in addition to producing greater amounts of β-caryophyllene, two other molecules are also increased: α-copaene and α-humulene. So we asked ourselves what their role might be,” explains Magnani.

Blend

According to Haroldo Xavier Linhares Volpe, another author of the study and an entomologist at FUNDECITRUS, when the plant emits this aroma, the insect receives not one molecule, but rather a mixture of these compounds in a ratio of 1 (α-copaene) to 100 (β-caryophyllene) to 10 (α-humulene).

“So we decided to study the other two molecules, either alone or in mixtures, following the original ratio, and found that α-copaene repels the insect at a dose 100 times lower than that required for β-caryophyllene. When it was studied alone, we were impressed and thought: we’ve found a much more potent molecule that can be prioritized in new repellent strategies,” explains Volpe. “As for α-humulene, we found that it’s neutral in this communication. α-Copaene and β-caryophyllene are enough to tell the insect to go away.”

The researchers also tested the repellent potential of copaiba oil diluted in a solvent (hexane) to see if it also worked, and the results were positive.

To ensure that the insects were exposed to the aromas in a similar way to what occurs in nature, the scientists developed and perfected a dynamic diffuser to disseminate the compounds in an arena where the insects are confined and can walk around and make choices about the aromas offered.

“The substances that make up the compound have different volatilization times, so we couldn’t, for example, soak cotton with the fluid containing the molecules and leave it in the arena. We used a small glass lamp, closed at the top, with a cotton wick that pulls the liquid from inside the bottle and releases it into the arena used to evaluate the psyllid’s behavior,” explains Professor Walter Leal, a researcher at the University of California, Davis, and a collaborator on the project. “With the help of this diffuser, we tested both copaiba oil and the molecules individually, as well as mixtures of two or three molecules.”

Leal points out that, for the first time, it was possible to simultaneously name and quantify the compounds emitted into the air that reached the insects thanks to the multi-user equipment obtained with support from FAPESP. The Foundation also supported the work through two Thematic Projects (17/21460-0 and 15/07011-3). “What matters to companies that develop repellent or attractant products is what goes into the air, not what’s put in the diffuser,” Leal points out. This information is valuable for developing products for the integrated monitoring and management of agricultural, veterinary, and urban pests.

Push-pull and kill strategy

Other host plants for the insect include curry and myrtle. “They’re good for the psyllid to multiply, but it can’t acquire the bacteria from curry at all, and only 1% of the insects that feed on myrtle acquire the bacteria,” says Volpe. The researcher also reveals that the idea is to use a scientifically proven, commercially available technology called “push-pull and kill.”

“We want to work with the orange tree association to repel the insect by attracting it to a bait plant, which could be curry, but we’ll have to kill the insects in the curry and we’re still studying how to make it lethal to the insect. We have a line of research on the subject and published articles proposing curry as an attractive plant,” he points out.

The article “α-Copaene is a potent repellent against the Asian Citrus Psyllid Diaphorina citri” can be read at www.nature.com/articles/s41598-025-86369-1.