Changing Psyllids to Make Them Incapable of Spreading HLB

Jim Rogers HLB Management, Research

By Kirsten S. Pelz-Stelinski and Lukasz L. Stelinski

Managing Asian citrus psyllids (ACP) remains a consideration for growers because psyllids transmit the citrus greening disease pathogen, Candidatus Liberibacter asiaticus (CLas), to newly planted trees. There are several tools that can reduce psyllid populations or mitigate the symptoms of HLB and improve the health of infected trees. However, additional methods that could help prevent transmission of CLas to newly planted trees could serve as an important complement. Replanted citrus groves could get into production faster if pathogen transmission could be prevented. Although ACP can be suppressed with insecticides, these tools are expensive and have undesirable consequences such as resistance and collateral death of natural enemies.

psyllids
A novel strategy to block transmission of the CLas pathogen by ‘gumming up’ the psyllid gut includes the following steps: A) CLas binds to the gut surface and enters before it can enter into the psyllid and be transmitted. The psyllid becomes a vector. B) Gut-binding peptides (GBP) were selected for binding to the ACP gut to outcompete the pathogen. C) GBP ‘gums up’ the surface of the gut and prevents CLas binding, thereby blocking the bacterium from entering ACP. GBP produced by Wolbachia in the gut of the psyllid will be tested for CLas transmission blocking.
(Graphic created with BioRender.com)

A potential new method for managing greening is development of non-transmitting ACP. Several bacteria, called endosymbionts, naturally live inside ACP. These bacteria cannot be transmitted to citrus and do not act as pathogens. They also cannot survive outside of the insect. The insect and bacteria mutually benefit one another.

WORK WITH WOLBACHIA
One novel approach for developing non-transmitting ACP is modifying one of the endosymbionts living in psyllids, called Wolbachia, to produce proteins that target the CLas pathogen after the ACP ingests it. The process of modifying naturally occurring Wolbachia to produce new proteins is called paratransgenesis.

Scientists have specifically investigated Wolbachia for many years as a potential tool to control spread of human-borne pathogens by mosquitos. Modifying this bacterium in mosquitos has been shown to reduce the transmission of several viruses among people with no adverse effects on humans or the environment.

The first step in this approach with ACP was isolating their specific Wolbachia. Based on the methods used with mosquitos, researchers established cultures of Wolbachia taken from ACP and grew them in large quantities. The second step was introducing genes into Wolbachia that would modify ACP in the desired way. Modified Wolbachia are then reintroduced into the psyllids by injecting young ACP. These modified Wolbachia produce new proteins inside the ACP that inhibit transmission of the CLas pathogen to citrus.

THE ROAD TO COMMERCIALIZATION
This project has met several technological milestones in the laboratory, including establishing a Wolbachia system. Certain challenges must be overcome before delivering a field-ready tool to commercial groves. First, researchers are still testing possible genes that would most effectively disrupt the pathogen. A goal of the current project is to identify peptides that compete with the citrus greening pathogenfor a limited number of binding sites located in the ACP gut. These peptides are intended to outcompete the pathogen and ‘gum up’ the psyllid gut, preventing the pathogen from colonizing the insect. Normally, such colonization must occur before the pathogen can be transmitted. Ultimately, this strategy would break the cycle of pathogen transmission at the source by neutralizing the psyllid.

A psyllid population that cannot transmit CLas would be mass-reared for release in commercial citrus groves to replace local ACP populations. The existing ACP population could be reduced locally with insecticides before releasing non-transmitting ACP. Any remaining resident ACP would mate with non-transmitting ACP, and the Wolbachia modified with the anti-pathogen protein would be passed to the offspring. Wolbachia stay in the ACP throughout its lifetime and are naturally passed to offspring.

In this way, the local population of ACP would be changed and no longer transmit the greening pathogen. Periodic releases of non-transmitting psyllids would be necessary to maintain this in the local population. Although this technology would not likely solve the greening problem, we expect it could serve as an important complement to current tools by reducing the need for broad-spectrum insecticides and facilitating biological control agents.

Acknowledgments: This project was supported by the Defense Advanced Research Projects Agency, award number D16AP00031, from the U.S. Department of Defense and the Emergency Citrus Disease Research and Extension Program, award number 2021-70029-36053, from the U.S. Department of Agriculture National Institute of Food and Agriculture.

Kirsten Pelz-Stelinski is associate center director and a professor, and Lukasz L. Stelinski is a professor, both at the University of Florida Institute of Food and Agricultural Sciences Citrus Research and Education Center in Lake Alfred.

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