A new strategy for combating the vector of the bacterium that causes huanglongbing, also known as greening, considered the most destructive citrus disease in the world and present in 17% of orange trees in São Paulo State, Brazil, can be developed from the discovery that three citrus plants produce an essential oil that repels the insect.
Brazilian researchers affiliated with the Federal University of São Carlos (UFSCar) and working under the aegis of the National Institute of Science & Technology for Biorational Control of Insect Pests (INCT-CBIP), funded by the National Council for Scientific & Technological Development (CNPq) and by FAPESP, analyzed the chemical composition of the essential oils produced by 22 species of the genus Citrus, including oranges, limes and tangerines.
Their purpose was to understand the effects of these substances on organisms that interact with the plants, such as Diaphorina citri, the Asian citrus psyllid. D. citri transmits Candidatus Liberibacter spp, including the bacterium that causes huanglongbing (HLB). The analysis was performed as part of the Thematic Project “Integrated studies for leaf cutting control”, also supported by FAPESP.
D. citri is only 3 mm long. It sucks sap from terminal shoots but does not cause significant direct damage. What destroys the plant is the bacterium transmitted by the insect’s eggs – a single female lays as many as 800 eggs.
The researchers discovered that the insect prefers to lay eggs on certain citrus species rather than others. Two in particular are unattractive: Citrus reticulata, known as the Murcott mandarin, and the Swingle citrumelo, a hybrid of C. paradisi grapefruit with Poncirus trifoliata, the trifoliate orange.
“The chemical composition of the volatile oils from 22 genotypes of Citrus and related genera hasn’t been properly explored until now,” said Maria Fátima das Graças Fernandes da Silva, a researcher at UFSCar’s Center for Exact Sciences & Technology (CCET) and INCT-CBIP’s principal investigator. “We were able to understand these substances in greater depth and explore their ability to attract or repel the psyllid using chemometrics, a set of mathematical and statistical methods for the analysis of chemical data,” she added.
Their findings include the fact that, above all, D. citri prefers Murraya paniculata, the orange jasmine, a native of Asia widely cultivated in Brazil as an ornamental plant. Chemical analysis of its essential oils confirmed the plant as the insect’s preferred host.
According to Fernandes da Silva, this plant has been eradicated from citrus-producing hubs because of the psyllid’s preference for it and its association with contamination of citrus groves.
“We now know the substance produced by the plant responsible for attracting the insect, as well as the oils produced by the citrus genotypes that also attract it, facilitating contamination,” she said.
The genotypes that least interest D. citri all share three compounds found only in their essential oils: phytol, (Z)-beta-ocimene and beta-elemene. The researchers concluded that these three compounds may act as repellents, making the plants less attractive to D. citri for ovipositing purposes.
The hypothesis that certain citrus plants might present some kind of resistance to D. citri arose at the Sylvio Moreira Citriculture Center in Cordeirópolis, São Paulo State, which had observed that the psyllid preferred some of the 22 species cultivated there, avoiding the rest. Researchers at the University of São Paulo’s Luiz de Queiroz College of Agriculture (ESALQ) identified the ones assumed to be unattractive to the insect and the group at UFSCar then investigated to identify which substances produced by the plants in question might make them repellent.
The researchers used gas chromatography combined with mass spectrometry to identify, separate and analyze the constituents of each essential oil in these plants.
To measure each oil’s capacity to attract or repel D. citri, they placed a group of psyllids in the bottom of an olfactometer, a Y-shaped tube containing a sample of oil in each exit arm, observing which oil was preferred on the basis of odor. Chi-square tests and other statistical techniques helped determine a repellency index.
“Attraction by oils from C. reticulata and the Swingle citrumelo was very low, leading us to conclude that their constituent compounds are in fact repellent,” Fernandes da Silva said. “This knowledge could be the basis for plant protection strategies.”
The researchers are now working on an analysis of the essential oils using electroantennography to measure the output of the insects’ antennae to their brains for a given odor, with the aim of discovering why the oils are repellent. Gas chromatography used in conjunction with this technique should enable them to identify precisely which molecule is responsible for repellency.
Citrus plants in Brazil are mostly grafted rather than grown from seed. Grafting involves attaching a twig (scion) from one tree to the stem of a tree seedling (rootstock). The scion becomes a permanent part of the tree over time. Scion and rootstock are usually different species. Grafting can be used to produce seedlings of plants that are difficult to reproduce or to combine characteristics of two species.
No commercial varieties of citrus scion or rootstock plants are HLB-resistant. Contaminated new plants do not even reach the production stage, and those already in production suffer from massive preharvest fruit drop and eventually die. The only effective method available to control the disease has been constant inspection and immediate elimination of plants with symptoms.
HLB originated in Asia and was identified in Brazil in 2004. The disease is present in all citrus-growing regions of São Paulo State. It is also found in Minas Gerais and Paraná, as well as other major citrus-producing countries, such as Mexico and the US. In Florida, Brazil’s leading competitor in orange production, HLB was first detected in 2005 and now affects 80%-90% of groves and about 70% of trees.
“This disease is a very serious problem for the citrus industry in Brazil and worldwide,” said Fernandes da Silva. “Entire groves have to be eliminated if a large percentage of trees are contaminated. Even trees without symptoms have to be destroyed to make sure no sources of contamination are left because they could spread to other trees and groves.”
The discovery that certain essential oils are repellent could be important to improve grafting. “In the absence of a plant that strongly resists HLB, it might be possible to produce grafts with plants that we now know to be repellent to its vector,” she said.
A table of repellency indices for each species analyzed is presented along with the other results of the study in “Essential oil variation from twenty-two genotypes of Citrus in Brazil – chemometric approach and repellency against Diaphorina citri Kuwayama,” published in the journal Molecules and available at mdpi.com/1420-3049/21/6/814. The authors are Moacir dos Santos Andrade, Leandro do Prado Ribeiro, Paulo Cesar Borgoni, Maria Fátima das Graças Fernandes da Silva, Moacir Rossi Forim, João Batista Fernandes, Paulo Cezar Vieira, José Djair Vendramin and Marcos Antônio Machado.