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13th March 2015
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Citrus greening

Jasmin Fox-Skelly, 13 March 2015

Citrus

Citrus orchards around the world have been stricken with citrus greening disease or, as it is known in China, huanglongbing (HLB), which translates as ‘yellow dragon disease’. Growers, agriculturalists and academics liken it to a cancer, with vast numbers of trees developing the characteristic deformed roots and misshapen fruits. Diseased trees never recover, usually dying three to five years after becoming infected. Yields from infected trees are poor, and the fruit mostly worthless owing to its small size, poor colour and bad taste.

In the US, the citrus industry in Florida, which provides up to 80% of US orange juice, has been hardest hit. Since the disease struck the state in 2005, 80% of trees and all of Florida’s commercial citrus groves have become infected, resulting in a market loss of more than $4.5bn in crops and nearly 8300 jobs. Now orchards in Georgia, Louisiana, Texas and California are at risk. Elsewhere in the world, the disease has killed 50m trees in Asia, and more than 10m trees in Africa. In Brazil, the world’s biggest producer of orange juice, 3m orange trees were destroyed in 2004, the year the disease was discovered.

The disease is caused by the bacteria, Candidatus Liberibacter asiaticus, which starts its journey in the tree’s roots where, after a period of incubation, it moves up the trunk, where it blocks the passage of nutrients to the leaves and fruit. Leaves turn yellow, and the oranges, deprived of sugars from the leaves, remain green and sour. The rapid spread of the disease can be attributed to a tiny insect, the Asian citrus psyllid (ACP). The psyllids pick up the bacteria when they eat leaves from infected trees, spreading it to healthy trees when they next feed.

There is currently no cure for HLB, and removal of infected trees and insect control are the main management strategies to limit or prevent its spread. Research is focused on killing the bugs and the bacteria that spread the disease; on increasing the immunity of trees; and on modelling outbreaks so that growers know which trees to target.

Killing the psyllids

Many pharmaceutical companies are focusing their research on controlling the pest responsible for spreading the disease. Caydee Savinelli, an entomologist at crop protection specialist Syngenta, comments: ‘Originally when the disease was first detected, growers began to take out the trees but they quickly realised that there would be no more trees left. What growers are now trying to do is help young trees become more established, help them grow and produce fruit – while keeping the insects from feeding.

‘Keeping the insects from feeding is key, because this is how the disease is transmitted. We have developed a neonicotinoid insecticide called thiamethoxam which does just that. Products that harm the psyllids but don’t stop them from feeding are futile as they will just carry on infecting trees,’ says Savinelli.

The problem with insecticides, however, is that bacteria can become resistant to them, making them less effective. ‘At Syngenta, we are working with researchers from the University of California, US, to develop a strategy where growers can switch from neonicotinoids to other pesticides, then switch back. This helps stop resistance,’ adds Savinelli.

Chemicals giant Bayer is also concentrating on tackling the bug vectors, with a year-round spray programme consisting of a broad range of insecticides. The company also collaborates with researchers at the University of Florida, US, on Admire Pro, a soil-applied systemic insecticide aimed at the citrus psyllid. In collaboration with the Citrus R&D Foundation, Bayer is also looking to develop compounds that target Candidatus Liberibacter asiaticus.

While commercial citrus grove farmers can look after their trees by spraying insecticides, in states such as southern California where there are more than two citrus trees per house, homeowners may be unwilling to buy these expensive products. There is a danger, therefore, that once the disease hits trees in the suburbs, it will spread to commercial groves.

In an attempt to prevent this happening in California, where psyllids are rife but there has only so far been one case of citrus greening, researchers are experimenting with other ways of controlling psyllid bugs.

For example, since 2012, researchers at the California Department of Food and Agriculture have been releasing parasitic wasps from Pakistan that attack the Asian citrus psyllids.1 The wasps, which are about a third of the size of the psyllids, lay their eggs on the bug’s stomachs. When they hatch, the larvae feed on their host, eventually killing them. Hundreds of thousands of these wasps, Tamarixia radiate, have been released so far, and in May 2014, the US Department of Agriculture promised the state funding of $1.5m to increase the amount to around a million wasps each year. They are hoping that the parasitic wasps will spread to citrus trees in the suburbs, thus protecting Californian oranges from the same fate as Florida’s citrus crops. Another wasp from Pakistan, Diaphorencyrtus aligarhensis, is also currently in quarantine and may soon be used to kill psyllids.

Meanwhile, other scientists are trying to exploit the citrus odours released by orange trees to trap psyllids. Two recent studies found that psyllids detect plant odours using tiny pit-like sensors on their antennae.2,3 Once the neurons in the antennae are activated by the smell, the psyllids go hunting for the source.

Anandasankar Ray, director of the Center for Disease Vector Research at the University of California, US, and his team identified a suite of odour molecules that activate these neurons. By mixing them together, the researchers developed an efficient attractant that could be used to lure the psyllids to yellow sticky traps. The traps caught nearly 230% more citrus psyllids than conventional traps placed on the same trees. The chemicals are relatively cheap, readily available in nature and safe for humans. Other odours that were found to block the psyllid’s olfactory system have the potential to be developed into repellents.

‘Further work is needed, but this research could lead to designs that attract the psyllids into a trap containing treatments like insecticides to kill them,’ says Ray. ‘Effective surveillance traps could also be used by the growers to check whether the psyllids are in a grove. This information could be used in decisions of insecticide use and other management practices. Additional tests on the trapped psyllids, such as the polymerase chain reaction (PCR), could inform state agencies whether they carried the disease,’ he added.

Killing the bacteria

Another way of tackling the disease is to kill the bacteria. For example, researchers led by Yongping Duan from the Agricultural Research Service in Florida, US, found that heating potted citrus seedlings in greenhouses kills the HLB bacterium, and can rid the seedlings of disease symptoms. The study also showed that the benefits can last for at least two years.

The infected seedlings were exposed to different levels of heat in growth chambers for periods ranging from two to 10 days. The researchers then measured how much of the bacterial DNA was left in the trees. Infection levels were measured a week before heat treatments began, and again 30, 60 and 270 days after they ended. The results showed that although constant exposure to high temperatures destroyed the foliage of citrus seedlings, if heat was applied intermittently then the leaves would survive, and HLB infection was reduced or even eliminated. Two years after their heat treatments, the seedlings have remained free of HLB and are producing normal fruit.

In a separate field trial, Duan found that heating HLB-infected trees in the sun by encasing them in plastic ‘tents’ could also prolong their productivity.4 The biological causes behind the impressive results are unclear but it seems that the heat kills some of the bacteria and weakens the infection ability of the remaining bacteria, thus prolonging the tree’s productive life.

Computer models

A problem facing growers is how to know which trees to target for spraying or removal – relying on visual symptoms does not work because trees often have the disease months or years before they show symptoms. Thus, many scientists are developing computer models that both predict the likelihood and extent of further spread of the disease, and measure the effectiveness of different strategies for disease control.

Stephen Parnell has been working on citrus greening for the past decade, first with the US Department of Agriculture (USDA) in Florida, and then at the UK-based Rothamsted Research Institute. Together with researchers at the University of Cambridge, UK, he has recently developed a computer model that can simulate the spread of the disease.5 The model can also be used to assess the economic costs and potential benefits of different control measures.

‘I use these models to test surveillance strategies for early detection,’ explains Parnell. ‘Early detection is crucial to have any chance of controlling the disease, at least in a cost effective way.

The model is currently being used by the USDA to conduct state-wide searches for HLB in Florida.

Genetic engineering

Another way of tackling the global spread of citrus greening disease is to increase the levels of immunity in citrus trees. This could be achieved by using genetically-modified trees that are resistant to the bacteria. GM crops are unpopular among consumers, particularly in the US, but many researchers and growers agree that it is unlikely that oranges will remain in Florida unless new, modified strains that are resistant to the disease are grown.

Erik Mirkov, a plant pathologist at Texas A&M University, US, is investigating genetically-modified oranges. In 2000, he began looking into whether citrus plants could be engineered so that they could resist bacteria.6 He originally looked to animals that naturally produced antimicrobial proteins, such as scorpions, honeybees and sarcophagus beetles. Although the science was promising, Mirkov was concerned that consumers would not drink orange juice that had been modified by beetle, or any insect, genes.

For GM foods to be accepted, he explains, the genes should preferably come from another plant that is already part of the human diet.

The spinach plant, which naturally produces several proteins that attack a wide variety of bacteria and fungi, was the ideal candidate. Incorporating the genes responsible for these proteins into a citrus tree, Mirkov reasoned, could make it resistant to a broad spectrum of diseases, including the newly discovered citrus greening disease. Mirkov and his colleagues began putting the spinach genes into the orange tree’s DNA, hoping to deliver the antimicrobial proteins to the innermost layer of bark, which is where the deadly bacteria disrupt the flow of tree’s nutrients.

His research is already showing promising results. By the second and third generations, trees containing the spinach genes that were grown in greenhouses crawling with psyllids, continued to thrive even after 16 months in the greenhouse. Field trials have also been successful, with a large proportion of second- and third-generation trees still healthy after almost two years. Mirkov is now on the fifth version of his technology, and is applying it to grapefruits, lemons, and rootstocks that growers use as a base for their trees.

In order to market the technology, Mirkov is working with Florida-based Southern Gardens, which claims to be the world’s largest supplier of 100% pure Florida not-from-concentrate (NFC) orange juice to the private label industry and major brands, to deregulate the technology.

This is, however, a long process that requires approval from the US Environmental Protection Agency (EPA), the Department of Agriculture and the Food and Drug Administration. If successful, the first commercial planting of the genetically protected citrus trees could take place in three to four years. However, whether the American public would accept orange juice genetically-modified with spinach genes is another question.

As one scientist, who wanted to remain unnamed, said in The New York Times: ‘People are either going to drink transgenic orange juice or they’re going to drink apple juice.’

References

1 H. Rosner, National Geographic Magazine, 17 June 2014.
2 A. Ray et al, PLOS One, 2014, doi: 10.1371/ journal.pone.0109236.
3 A. Ray et al, Chemical Senses, doi: 10.1093/ chemse/bju023.
4 D. O’Brien, Agricultural Research Magazine, 2013, 61(7), 4.
5 S. Parnell et al, Ecological Applications, 2014, 24(4), 779.
6 T. E. Mirkov et al, Phytopathology, 2005, 95, 6.

Jasmin Fox-Skelly is a freelance science writer based in Cardiff, UK

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