MOLECULAR BIOLOGIST Robert Shatters, center, postdoctoral student Kent Morgan, right, and technician Erica Egan, are working on several facets of citrus greening disease. They are at the U.S. Horticultural Research Laboratory at Ft. Pierce, Fla.
Entering the U.S. Horticultural ResearchLaboratory feels like walking through the front door of a blended mixture of a large urban hospital and a university library.
Although it is whisper-quiet, the people inside move with a quick urgency, hustling on unnamed but apparently important missions.
Situated on a piece of unexceptional land between I-95 and Florida’s Turnpike west of Ft. Pierce, Fla., many of the 150 USDA/Agricultural Research Service staffers and scientists working here focus on saving the citrus industry from destruction by citrus greening disease.
They realize they’re in a race against time. They think they are edging ever closer to winning.
“Citrus greening is the overwhelming major research thrust of this organization,” says Calvin Arnold, director of the laboratory. “Eleven of our 29 scientists work exclusively on greening, and under them are post-docs, support scientists, visiting scientists and technicians.
“Our approach is what we call a three-legged stool. One leg is the citrus tree itself, where it might be possible to breed natural resistance. The second leg is the pathogen, the disease-causing organism. The third leg is the vector, the citrus psyllid.
“Knock any leg out and the stool falls. We have a strong research program in all three areas. If any of the three approaches work, then we beat the problem.”
Some citrus industry leaders think the lab’s scientists are on the right track and could come up with an answer to the greening threat in a few years.
“They have some of the world’s brightestminds in citrus working there,” says Doug Bournique, executive vice president of the Indian River Citrus League located a half-hour drive up I-95 on the outskirts of Vero Beach. “They went out and hired the best people they could find, and I think it is going to pay off.”
Arnold says, “We’re looking for some way we can intervene and break the life cycle of the Asian citrus psyllid. It’s a sucking insect, and if we interfere with either its feeding or breeding cycle, we can stop it. One really good breakthrough could be a major factor in turning this thing around.”
Even though the greening problem is intense and growers want a solution as soon as possible, the researchers here carefully observe proper scientific technique. They may be moving quickly — but not in a headlong rush.
“Research is expensive and takes time,” Arnold says. “It’s slow; it has to be randomized and replicated. We’re doing statistically sound experiments. When we lose credibility as a research organization, we might as well lock the door and go home.”
Using a magnetic key card to open doors to the inner sanctum of the laboratory where the scientific work takes place, he notes that the researchers are organized into four groups, each headed by a leader functioning as an administrative officer.
“They report to me, so I’m able to provide overall communication and assess where we’re going as an organization,” he says.
“We work closely with allthe regional citrus groups, Florida Citrus Mutual, and other agricultural groups like the Florida Tomato Committee and the Florida Fruit and Vegetable Association. Our citrus work gets $3 million in funding from the industry, so we keep the growers apprised of what we’re doing.”
Arnold heads down a hallway and stops to talk with Yong Ping Duan, a bacteriologist trying to find ways to fight the bacteria that causes greening disease.
“I’m especially looking at the virus inside the bacteria, the phage, we call it. It is a type of virus. A phage could be beneficial to a bacteria, or it could attack it,” Duan says.
“We are getting more knowledge about this virus, how it affects the bacteria and how that affects the disease.”
Duan discovered that the citrus greening bacteria has difficulty surviving at fairly high temperatures and thinks that weakness might be used to stop it.
“When it is kind of warm, 40 degrees Celsius, the bacteria has difficulty. The problem is, we can’t control for consistent temperatures in trees in commercial groves.”
That failed to stop Duan, however. He put a small portable greenhouse-type structure over a citrus tree in his back yard that is infected with the greening disease. It trapped heat and effectively stopped the disease. Duan’s tree, which he says was very sick, recovered and quickly put on new growth.
“We’re going to do some controlled-environment trials in groves now and try to improve our results,” he says. “It’s a challenge to put it in the field. We’re going to try it in conjunction with heat treatments and antibiotics. So far we have identified two antibiotics that are very effective on the disease, but are not approved in citrus.”
The heat treatment conceptwould probably work best on young trees, Arnold says. “We can try it with young plantings where we see the infection before it is widespread. It could be a very good treatment for home growers.
“Just the solar heat from the sun inside that portable greenhouse is enough. No FDA or EPA registration is required.”
Duan’s work leads him to believe the citrus psyllid, vector of greening disease, and the disease itself arrived in Florida separately.
“It is the result of multiple introductions into Florida. We think the psyllid arrived in 1998. The disease probably arrived later. When the two came together the problem started.”
The notion that the disease and psyllid came into the U.S. from Asia through the Miami airport and spread from there could very well be incorrect, the lab’s scientists say.
“Our psyllid probably came from the Middle East,” says Wayne Hunter, the lab’s lead scientist working on the psyllid genome project. “It moved along until something brought it to the U.S. We don’t know where the entry point was.”
An entomologist by trade, Hunter says the DNA of a virus found in the psyllid’s gut, Wolbachia endosymbiont, shows it did not originate in Asia. “There are different Wolbachia populations in China and the Middle East. Ours is from the Middle East.”
That seemingly minor point could prove to be key in fighting greening disease.
“The climate is drier in the Middle East, and less diverse than Florida. There are different parasitoids, and they grow citrus in a different manner.”
Hunter agrees with Duan that the disease itself originated in China. “What we have is the introduction of the insect from one source and the disease from another source. The psyllid probably moved in on plant material, though we aren’t sure of that. Two years after the insect, the disease was probably accidentally introduced. Then the psyllid propagated it.”
The lab’s work on the psyllid genome is groundbreaking. “It’s the first psyllid ever done,” Hunter says.
“We are looking at active genes in the psyllid. You have to have that for gene disruption. That could help us find something specific to control the psyllid. We don’t want to kill beneficial insects. I should emphasize that what we’re looking at here is natural, not transgenic.”
Four research labs are workingon delivery mechanisms that might defeat the tiny insect. “Growers want something they can spray,” Hunter says. “This project is designed so we don’t have to change the behavior of the industry. Look at what they currently do — they spray.”
Even so, the scientists, being an ever-curious bunch, are investigating other delivery methods for a control product. Applying it through irrigation systems might work. So could applying it through patches similar to nicotine patches humans use to try and stop smoking. Workers could slap a patch on each tree and be done with it, no spraying involved.
“Our treatment goes to the tree’s xylem and phloem, an approach that is very good for a plant system. It is specific to the insect,” Hunter says.
“We’re identifying genes that might be the psyllid’s weak point. The material we’re putting in the xylem and phloem would prevent development of something the psyllid needs to survive, like wings. This is the whole future of insect pest management.”
The process, called RNA interference, or RNAi, affects the activity of specific key genes within the organism. Once known in the scientific community as gene silencing, it effectively turns genes on and off.
“It can be used in very, very specific ways,” Hunter says. “What we want to avoid is spraying in the groves and killing everything, because a lot of beneficial insects out there help growers. It will let us do a better job of integrated pest management.”
Hunter’s group of researchers continues investigating the psyllid, looking for weak genetic points.
“We’re using cell culture to see the virus in it. We can take out its RNA and DNA and sequence that in pieces, then compare it to a database to see which virus this is related to,” he says.
“It is very important to find the viruses. We have discovered 11 viruses from different insects.”
Hunter’s team identifiedfour previously unknown viruses in the psyllid’s gut. “We don’t know what these new bacteria do within the psyllid,” he says. “Everything about it is very interesting — and very complex. If you consider it mathematically, there are 10,000 potential variables within the psyllid. Our work is cut out for us.”
It remains possible that a predator insect or parasite could help solve the Asian citrus psyllid problem.
“When populations get out of control, pathogens take advantage of that,” he says. “Predators typically take three to five years to adapt to that as a food item. We’ll see what happens out there in the citrus groves.”
Researchers might also be able to tweak the citrus tree itself in order to generate natural genetic resistance to greening disease.
“We’re now able to identify a couple of varieties with classical based defense responses to it,” says Robert Shatters, a molecular biologist leading one of the lab’s groups working on the problem.
“These varieties are not immune, but we do see differing degrees of resistance. One of these is resistance found in sour orange. We may be able to put that into susceptible rootstock for grapefruit. It’s not difficult to make a transformation. We’re really excited about that.”
The psyllid only lives on citrus. One of its weak points, Shatters thinks, could be its sheath, a straw-like structure it sticks into the tree to suck up fluid.
“It’s kind of like a drilling well. Beneficial insects don’t do this. Aphids, whiteflies, scales and psyllids do. If the psyllid can’t get to the vascular tissue of the tree, it can’t pick up or distribute the greening disease. We’d like to block its ability to pick up or transfer the disease,” he says.
“We developed a study on how the psyllid makes these sheaths and found an inhibitor to the process. In that case, the psyllid’s saliva never solidifies and hardens in the tree, so the disease would not affect the tree. Our hope is that we can find a way to do that in the field.”
That might be done with a chemical application or with a gene inserted into the tree itself.
“We want the psyllidto leave before it infects the tree,” Shatters says. “The bacteria only grows in the tree’s phloem. Anything we can do to disrupt it, to make it feel uncomfortable, where it will leave before getting the bacteria to that area will keep it from spreading the disease. We have found an inhibitor that works.”
Shatters says the Torrey Pines Institute for Molecular Studies at Port St. Lucie, Fla., is working with his group on the project, studying ways to block the psyllid.
“They have a special chemical that looks promising. They also have DNA libraries and methods for screening trillions of molecules in hundreds of assays.”
Zeroing in on the Asian citrus psyllid is simplified a bit by the fact that its diet is limited to citrus. Adults live for only a couple of months. After hatching, the young go through five nymphal stages. If researchers can find a weakness at any point, greening disease can be stopped.
“If it can’t survive long enough to complete its life cycle, then it can’t spread the disease,” Shatter says.
He worries that growers currently fighting the psyllid with general insecticide sprays may do long-term harm to the industry.
“I understand why they’re doing it, but heavy insecticide use is not compatible with how we want to control this insect. We’re keeping the population down with an insecticide but that also keeps us from doing anything else,” Shatters say.
“The big potential problemis that the psyllid could develop resistance to the insecticide. If that happens, there is nothing else in our arsenal. Then, it’s just a question of when, not if, insecticide resistance pops up. What we have to do is develop integrated pest management strategies so we don’t mess up the whole citrus ecosystem.”
Arnold, the lab’s director, has four decades of experience as a scientist in the U.S. and Africa. He grew up at Okeechobee, about an hour’s drive west of here. His family was in the beef cattle business, but he spent his career working on horticultural projects. He came to the Ft. Pierce USDA-ARS lab after retiring from the University of Florida, with stints at experiment stations in several locations.
Walking the lab’s hallways, chatting with scientists and technicians alike, Arnold seems like a football coach urging his team on to victory against a tough opponent.
“These are some really fine people,” he says. “They’re working hard and putting everything they have into it. They know what’s at stake for growers and how important agriculture is to Florida’s economy.”