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Title: Non-transgenic RNAi technology to control insects on citrus

Author
item ANDRADE, EDUARDO - Embrapa-Labex
item Hunter, Wayne
item Hall, David
item RIBERIO, CAUE - Embrapa-Labex
item ASTUA, JULIANA - Embrapa-Labex

Submitted to: Florida Scientist
Publication Type: Proceedings
Publication Acceptance Date: 3/7/2014
Publication Date: 5/3/2014
Citation: Andrade, E.C., Hunter, W.B., Hall, D.G., Caue, R., Astua, J.F. 2014. Non-transgenic RNAi technology to control insects on citrus. Florida Scientist (78)(1):5. AGR-O01.

Interpretive Summary: This research demonstrates a non-transgenic delivery application method for ribonucleic acid (RNA) interference strategies aimed at stopping the spread of citrus greening disease, known as Huanglongbing. The disease is caused by a bacterium, that’s transmitted by a tiny insect, called the Asian citrus psyllid. We designed dsRNA molecules based on the specific gene targets in these insects, which when ingested caused increased insect mortality. The dsRNA were administered in solution to plant cuttings, and then insects were placed on the cuttings. We observed insect mortality beginning on the fifth day after feeding on treated plant cuttings. The mortality rate varied between 46-90% in accordance to the specific transcript target selected. The results obtained in this research shows there is potential to build a strategy based upon RNAi for insect pest management against the Asian citrus psyllid.

Technical Abstract: This research demonstrated a non-transgenic delivery method for ribonucleic acid interference, RNAi, that reduced fitness as measured in increased mortality over time, of two insect pests of citrus, ie. psyllids and leafhoppers. The Asian citrus psyllid transmits a deadly plant-infecting bacterium of citrus trees that causes citrus greening disease, known as huanglongbing. Huanglongbing restricts the production and sustainability of citrus crops. Ingestion of a double-stranded ribonucleic acid, dsRNA, designed to multiple messenger transcripts specific to either the Asian citrus psyllid (Diaphorina citri); or glassy-winged sharpshooter (Homalodisca vitripennis) induced RNA interference down-regulates the specific messenger RNA in the insect. RNAi, is a naturally occurring cellular process that recognizes dsRNA, and cleaves the corresponding RNA strand, thus we designed dsRNA’s to the insects’ transcripts resulting in the down regulation of the targeted messenger RNA, which prevents production of the corresponding protein, thereby reducing the biological processes and fitness of the insects. The treatments were administered to insects using a cut plant feeding assay, at 100ng dsRNA/0.25g of plant tissue. Each of the trials was comprised of controls and replicated treatments of three cages each transcript, with 15 adult psyllids or 6 leafhoppers per cage. DsRNA green fluorescent protein was used as a non-target control. Observations of mortality were recorded daily for 17 days, with significant mortality beginning on day 5. Mortality rates reached 46-90%. Only healthy looking plant cuttings were used in the experiment. Trials showed the dsRNA treatment induced significant insect mortality over controls. RNAi approaches continue to show potential as a means of insect pest management.