News - April 30, 2015

A jacket to stop the bleeding

Roelof Kleis

Many of the chestnut trees in the Netherlands are bleeding. The cause is a bacterium which is hard to combat. Wageningen scientists have now found a solution that is as simple as it is ingenious: heating the trees.

A snugly wrapped up horse chestnut might look a bit odd, with a few metres of its trunk wrapped in a kind of blanket kept in place with a rope. But this is not an art project, it is science. That becomes clearer when the tree is ‘unwrapped’. On a Tuesday morning on a country estate in the Brabant village of Duizel, André van Lammeren (Laboratory for Cell Biology) and André Korsuize (PPO Flower Bulbs, Lisse) helped a chestnut tree out of its temporary jacket. The canvas hides a layer of bubble wrap and some radiator foil. ‘A thermal blanket,’ is what Van Lammeren calls the jacket. The actual heating is under the jacket: a yellow garden hose with warm water, coiled around the tree trunk.

So has the tree been cold? No, black spots on the trunk betray another problem. The spots look like blood clots. On trees further down the avenue, you can see even more clearly why the disease is called bleeding canker. From small splits and tears in the trunk run several slow trickles of reddish brown fluid. The disease was first reported in 2002, explains Van Lammeren. ‘It started in the west of the country and then spread south and east. It crossed borders and now chestnuts are infected in Denmark, Germany and Belgium too.’

In the Netherlands alone, a rough estimate by Van Lammeren suggests that 100,000 chestnut trees are affected. ‘That estimate is based on 400 municipalities and 500 trees per municipality. That comes to 200,000 chestnuts, so if half of them are affected that makes 100,000 trees. But I think that may be on the low side.’ On the estate in Duizen alone there are 160 chestnut trees, of which at least 60 are affected. The cause of the bleeding disease was only identified in 2006. It turned out to be the bacterium Pseudomona syringae pathovar aesculi, a variant which specifically affects the horse chestnut. How the bacteria came here is not clear, says van Lammeren. His guess is that it was imported from India via shipping. Nor is it known quite how the bacterium infects the tree. ‘But the infection route certainly goes through cracks and tears in the bark. The bacterium then destroys the bark fibre, so that sugar transport is no longer possible. The characteristic wound fluid is a by-product of this process. In the end the tree gives up the ghost.’

Once the culprit was identified, efforts could be focused on finding a way of fighting it. But none of the methods investigated led to a satisfactory result. Not, at least, until lab tests showed the bacterium to be sensitive to heat. Van Lammeren: ‘The bacterium turned out to stop growing at temperatures above 39 degrees centigrade.’ Then the idea came up: couldn’t we get rid of it through heating?’ And the answer, subsequent lab tests suggested, was yes. Jeroen Keijzer, a Master’s student at the time and now a Cell Biology PhD candidate, published an article about this in PlosOne in June 2012. Since then, his project managers Van Lammeren and Fons van Kuik (PPO Fruit, Lisse) have been working on proving that the method works outside the lab as well. In the trees in Duizel for example. In the past year and a half, trees have been wrapped up warm from Zwolle to Boskoop and from Haarlem to Duizel. Each treatment takes at least two days. In the lab two days at 39 degrees proved to be sufficient to kill the pathogenic Pseudomonas. Whether that works in practice remains to be seen. Master’s student Otto van der Linden is testing samples of bark from before and after the treatment for the presence of the pathogenic bacterium (see box).



Definitive proof that the heat treatment works will be delivered in the lab in the near future by Otto van der Linden. A nice job for the first-year Master’s student of biology. In simple terms, he has to prove that the pathogenic Pseudomonas bacterium is present in the bark before treatment and not afterwards. That proof is delivered using genetics. Fragments of DNA, known as primers, are identified which are specific to the bacterium. The primers function as a kind of fingerprint with which the presence of the bacterium can be proven. But first you must get hold of that bacterium. To do so, Van der Linden chops up the bark and soaks the bacterium out of it. But it goes down fighting. ‘The bacterium makes itself a kind of shell of polysaccharides to defend itself against the tree’s immune system,’ explains Van der Linden. ‘It infects the plant from inside that shell. There is a protocol but I am still working on perfecting it. Of course we want to be certain that we don’t miss any bacteria.’ The extract from those first few steps is then kept on a slide to grow. An antibiotic is added which kills many species of bacteria but leaves the pathogen alone. The relevant bacteria colonies are then tested genetically using primers. The first colonies are currently growing.

The temperature during the process is crucial, so sensors in the bark take the tree’s temperature continually. Through wifi, Van Lammeren has recently been able to track the temperature of the bark, the water and the air on his tablet in real time. A tree which absorbs water from the soil cools down as a consequence. That cooling must be compensated for; otherwise the bacterium escapes its demise. To date, all the treated trees are still alive, reports Van Lammeren. The trees are monitored regularly. But he cannot offer the participating tree owners – mainly municipalities and a few private owners – any guarantees yet. Nor is there any guarantee that the bacterium will stay away after treatment. ‘People who have had one cold can still catch another one. The same goes for trees,’ explains Van Lammeren. ‘The bacterium can return. But it is possible that the tree may develop some immunity. All these are things we don’t know yet.’ One thing is certain, and that is that at the moment heat treatment is the only method that appears to work. It does involve a lot of work, though. It takes the two men almost a day to help two trees in and out of their jackets. That will have to get faster and more efficient, acknowledges Van Lammeren. ‘But we are not that far yet. Commercial parties are interested in our method. But first we’ve got to prove it works. Only then can you take it further.’

Photo's: Roelof Kleis