The fight against a devastating citrus disease

Car licence plates in Florida have a picture of an orange displayed prominently in the middle. This US state is inextricably linked with orange growing: Florida has tens of thousands of hectares planted with row upon row of citrus trees. In 2021, the sector employed more than 30,000 people and was worth 7 billion dollars.

But the cultivation of oranges and other citrus fruits has collapsed in the United States. Production of citrus fruits in Florida is down by 90 per cent compared with 2006. ‘Over the past few years, we have gone from 150 million boxes of oranges per year to 10 million at most,’ a gloomy young grower told a local TV broadcaster in a news report. ‘Every time I drive along here, I see another abandoned orchard.’

There are various reasons for the decline in Florida’s orange production, but the main factor is the devastating citrus greening disease HLB. ‘The moment you see that disease in an orchard, you can assume a 70 to 80 per cent reduction in yields,’ says bacteriologist Jan van der Wolf. He carried out research at Wageningen Plant Research on the bacterium that causes HLB from 2020 until his retirement in April 2025. He and his team want to gain an understanding of the bacterium behind this citrus disease.

HLB stands for huánglóngbìng, a Chinese word that literally means ‘yellow dragon disease’. Chinese farmers first encountered the disease back in the 19th century. China has now cleared more than 100 million infected citrus trees, and over 56 million have been taken out in Brazil. Van der Wolf and his colleague are collaborating with scientists in Ethiopia, where cultivation is also badly affected by HLB. So far, HLB has not been found in Europe, but citrus growers in southern Europe are very worried. Spain produces almost six million tons of citrus fruits a year, and Italy nearly three million tons.    

‘There is a real danger the disease will reach here, transmitted for example by tourism or commercial traffic,’ warns Van der Wolf. That is why the Netherlands Food and Consumer Product Safety Authority has arranged government funding for the research at Wageningen — after all, one of its tasks is to protect public health by preventing the spread of diseases to the Netherlands and the rest of Europe. 

HLB is caused by a tiny, almost invisible enemy, the bacterium Candidatus Liberibacter, which is spread by psyllids, also known as jumping plant lice. The psyllids suck on infected plants, thereby ingesting the Liberibacter. The bacterium gets into the psyllid’s salivary glands and can infect the following plant through that route. ‘The bacterium can spread very quickly from one crop to the next because it is transmitted by flying insects,’ explains Van der Wolf.  

But the disease progresses slowly: ‘It can take months or even years for the symptoms to become visible,’ says Van der Wolf. ‘That makes it much harder to work out which trees in a field have been infected and should be removed. On top of that, it’s not always easy to recognise the symptoms. For example, you see stunted growth, yellowing leaves and “rabbit’s ear”, which is when branches stick out oddly. But some of the symptoms could point to other diseases as well, or a lack of minerals.’

The fruits of an infected plant cannot be used because the juice is too bitter. The fruits can also be hard and deformed. The fruits remain green at the top rather than turning orange, which is how the disease gets its common name of ‘citrus greening’. 

There is almost nothing that can be done to stop it. ‘Once a plant is infected, it can’t be saved,’ says Van der Wolf. ‘So growers try to prevent infection in the first place. They spray the crops with insecticide to combat the psyllids. They also look out for early signs such as yellowing leaves, stunted growth or deformed branches, then remove the infected plants as soon as possible to stop the disease from spreading further.’

The Liberibacter is a strange bacterium. ‘It is actually lame,’ says Van der Wolf. ‘The average bacterium has 5 million base pairs — the building blocks of DNA — but Liberibacter only has 1.2 million. Liberibacter species are highly specialised, targeting only one plant, and lack all kinds of properties. They are very selective in what they need in terms of genetic material. Perhaps that’s why the bacterium is so successful.’

Research into this specialised citrus killer is essential to protect the cultivation of oranges from HLB, but it is a difficult challenge. The bacterium is so specialised that it can’t survive without its host — and therefore doesn’t survive on a Petri dish either. That means that unlike other bacteria, Liberibacter can’t be cultured for research. ‘That would be so helpful, for example for investigations into fighting the disease and the resistance of plants.’ 

Van der Wolf first started investigating Liberibacter in 2020. ‘To begin with, I did wonder what on Earth I’d got myself into. I couldn’t even culture the bacterium and I’d never worked with it before.’ 

But the research at Wageningen has come further than Van der Wolf had expected thanks to the development of new DNA techniques. ‘We’ve got hold of so many fun toys over the course of time. The latest sequencing techniques let us analyse all the DNA of the plant material or insect in one go, for instance.’

Lina Russ has taken over the HLB research since Van der Wolf’s retirement. She knows exactly how to make good use of these new techniques. As a bioinformatics expert, she focuses on analysing bacterial DNA using advanced sequencing methods. ‘Sometimes it feels like you’re solving an enormous genetic puzzle,’ she says, ‘and you just hope all the pieces will be present.’

The first step was to obtain some suitable material, because Liberibacter is not found in the Netherlands. She was sent plant samples from growers in Ethiopia, among other places. In the lab, the DNA is extracted from the plant sample and decoded by a sequencer, which turns it into digital data. The computer then analyses the raw data and arranges the DNA fragments in the correct order. If there is enough genetic material for Liberibacter, Russ can reconstruct the bacterium’s genome.

But that is easier said than done. ‘We can’t culture the bacterium so we have to use material from plants that are infected with it and extract its DNA from that. That material includes not only DNA relating to the plant and Liberibacter, but also that of numerous other microorganisms. The trick is to extract the sequences relating to Liberibacter from that mix. We do have advanced techniques for separating out organisms, but some organisms look a lot like one another, which means you can’t always be sure whether you have extracted the right sequences or whether you have a complete set. That makes it difficult.’

Another method Russ uses a lot in her research is reference-based mapping, in which DNA fragments are compared with known genomes in databases. ‘You are looking for recognisable pieces in an existing puzzle,’ she explains.

To find unknown pieces as well, Russ also uses a technique called de novo assembly, which lets you reconstruct a genome without having to use a reference genome. Instead, the computer looks for overlap between fragments and slowly builds up longer sequences. ‘Then the real hard work starts. It’s as if you have 10,000 puzzles — each for a different organism — and all the pieces have been thrown into one pile.’ 

If Russ and her colleagues can manage to put the pieces of the Liberibacter puzzle together, they could add that new information to a public database. It now has the data Russ was able to obtain from the Ethiopian plant samples. By sharing this information, she can help combat the bacterium. Van der Wolf: ‘All we can do for now is prevent infections and remove diseased plants. Good identification and detection methods are needed for this.’

The Wageningen research is also helping in efforts to determine the genetic diversity of Liberibacter. Plant breeders need an understanding of the diversity to make it possible to select plants for resistance. That is because the solution in the longer term will have to come from plant breeding, thinks Van der Wolf: ‘Plant breeders have been working on developing resistant plants for a while, but it takes time before you see the results. Citrus trees grow relatively slowly and it can be ten years before you get the first fruits.’

It could be years before scientists really get a good understanding of Liberibacter. Citrus fruit prices are likely to rise worldwide in the meantime, think Van der Wolf and Russ. Citrus growers will probably also use insecticides more and more, with the inevitable consequences for the environment. Van der Wolf: ‘In countries where they have a lot of problems but not much money, such as Ethiopia, this is really threatening people’s livelihoods. I hope our research can help them.’