Since the introduction of Huánglóngbìng (HLB--yellow dragon disease--or better known as citrus greening disease) reared its ugly head on U.S. soil in a Florida citrus grove in 2005, the disease has been a major threat to commercial citrus production across the country.

Before arriving in North America, HLB had already carved a path of destruction across the Far East, Africa, the Indian subcontinent and the Arabian Peninsula, and was discovered in July 2004 in Brazil. In its wake it left citrus growers around the world astounded at the inevitable and long-lasting risks the disease poses to global citrus industry.

During the first couple of years after reaching Florida, the disease had destroyed a huge section of the state's successful citrus industry, and by 2009, just five years after its introduction in the region, almost every county within Florida had confirmed HLB cases among both commercial and private citrus groves. From there the disease spread to adjoining states, eventually reaching citrus growing areas in Texas and finally as far west as California.

The fight against HLB and the tiny psyllids that carry the bacteria from tree to tree is about as old as the disease itself. Recognizing the disease had the ability to threaten the global citrus industry, researchers from around the world began working on possible solutions to combat the spread of this dangerous citrus killer.

In spite of early efforts however, the tell-tale signs of the disease kept spreading.

The early symptoms of HLB include leaves with yellowing veins appear along with asymmetrical chlorosis referred to as "blotchy mottle." These are the most diagnostic symptoms of the disease, especially on sweet orange. Growers, ever fearful the disease would reach their trees, have been on constant lookout for leaves that are slow to develop and often with a variety of chlorotic patterns that often resemble mineral deficiencies such as those of zinc, iron, and manganese.

Regardless of treatment efforts, once established in a grove, the end result of the disease is proving to be inevitable, the complete decay and destruction of all infected trees.

Detection of the disease is one of the first hurdles facing citrus growers in modern times. When it comes to fighting HLB, growers face a number of unique challenges. For one, HLB-infected citrus trees do not show symptoms during the first year of infection, so there is a long period of time when a grower cannot visually detect an infected tree. But that hasn't stemmed research efforts.

The spreading pandemic of the disease served to rally the global citrus industry and the many researchers who support it. Soon new and innovative treatments were being tested. In addition to antibacterial management and control and management of the psyllids that carry the disease, tree removal became a standard procedure to help curtail the rapid spread of the bacterium.

Soon, beneficial parasitoids were introduced and widely used to help control psyllid populations. Heat treatments in nurseries and on field trees covered by plastic wrap offered some slowing of the disease process in early research efforts. Hundreds of millions of dollars were being spent worldwide searching for a cure to the disease. A zinc-based bactericidal spray seemed to offer some hope.

Before long, breeders were offering new citrus varieties that were proving resistant to the bacterium that causes HLB. Bio-engineers have been devising methods to make citrus trees less attractive to the psyllids that carry the disease. But in recent months a new idea has surfaced, and while no one is ringing the bell of victory, researchers on the project are quietly voicing new hope in the war against the disease.

HOW IT WORKS

According to researchers, the reproductive and feeding habits of the psyllid make it the perfect carrier of the bacterium. An infected psyllid creates a localized infection when it feeds and transmits the bacterium into a citrus tree. It does not take long for the bacterium to spread throughout the plant, but the inoculum is first concentrated in the leaves and stems where the infected psyllid feeds. Female psyllids lay eggs in the same region where they feed. If these females are infected, their nymphs, which begin feeding in the infected area of the tree when they hatch, eventually acquire the bacterium, molt to the winged adult stage and disperse taking it along with them.

So researchers at the Boyce Thompson Institute, a premier life sciences research institution located in Ithaca, New York on the Cornell University campus, have concentrated their recent efforts on the psyllid itself as a possible link to the control of the disease.

Michelle Cilia, a Research Molecular Biologist at the USDA Agricultural Research Service and Assistant Professor at the Boyce Thompson Institute (BTI), and her team of researchers have been looking at a protein that makes the bellies of citrus psyllids blue and the possible connection it may have with the natural process of spreading the devastating bacterium in the first place. Researchers say Asian citrus psyllids with blue abdomens have high levels of an oxygen-transporting protein called hemocyanin.

According to Cilia, the hemocyanin protein is commonly found in the blood of crustaceans and mollusks. When harboring the bacterium Candidatus Liberibacter asiaticus ( or CLas) the disease is spread by the Asian citrus psyllid. This bacterium force the psyllids to ramp up their production of this protein. Cilia lab scientists, along with colleagues at the University of Washington and the USDA ARS at Fort Pierce, Fla., identified important protein interactions that must occur to perpetuate the transmission of bacterium to new trees.

They examined interactions occurring between the psyllid and the bacterium, and between the psyllid and its beneficial microbial partners. They also compared protein expression levels in both nymphs and adults. Their research shows that adult psyllids appear to mount a better immune response to CLas as compared to nymphs, which may explain why psyllids must acquire CLas during the nymphal stage to efficiently transmit CLas once they become adults.

"For many decades, scientists lacked the ability to look inside insects that transmit plant pathogens and understand what is going on," said Cilia. "This is no longer true today, thanks to the painstaking work of our collaborators in the Bruce and MacCoss labs at the University of Washington. The new molecular tools developed by our University of Washington colleagues enable us to dissect the vector-pathogen relationship piece by piece to determine which components are important for transmission."

The group showed that hemocyanin interacts with a CLas protein involved in a vital microbial metabolic pathway called the acetyl-CoA pathway. Scientists have previously targeted this set of biochemical reactions in bacteria when developing antibiotics.

John Ramsey, a USDA ARS postdoctoral associate in the Cilia lab and first author of the study, suspects that the increase in hemocyanin, and the blue color it imparts to the abdomen, could be evidence of an immune response to CLas infection. The findings raise the possibility that this response could be harnessed to help control the bacterium's spread.

"The study is allowing you to look at your population of insects and say something about the immune system of the insect based on its color," said Ramsey. "There's the possibility that this could be a useful part of grove surveillance."

In future work, the Cilia group plans to test whether there are differences in each color morph's ability to spread the CLas bacterium. Results from this study will help inform future strategies to control citrus greening disease. Depending on which proteins they decide to target, these new approaches could prevent the psyllid from transmitting CLas or trigger an immune response against the bacterium.

This approach to controlling citrus greening, by blocking bacterial transmission by the psyllid, runs contrary to existing 'kill the insect' strategies, said Ramsey. Such an approach may provide a longer lasting solution because the insect isn't under pressure to evolve to survive the treatment, which commonly occurs with pesticide usage.