Benzoxazinoids (BXs) are substances that are formed in certain plants. They have an ability to counteract attacks from insects, parasites, fungi, and bacteria. BXs are found in different grass species, e.g. crops such as maize, wheat, and rye. The latter have very high concentrations of BXs. As part of his PhD project, Md Maniruzzaman Sikder from the Department of Agroecology investigated whether BXs play a role in the defence of maize plants against plant parasitic nematodes.

"Research has previously been done on how substances such as BXs play a role in plant defence, but it has not previously been investigated exactly what role the substances have on the composition of nematode communities," says senior researcher Mette Vestergård Madsen from the Department of Agroecology at Aarhus University. She and Professor Mogens Nicolaisen are supervisors for Md Maniruzzaman Sikder.

Good and bad nematodes

Nematodes can be both useful and harmful. Some nematode species benefit from metabolizing the remains of dead plants and animals in the soil, and they can also kill certain insects that are harmful to plants. But other species have a much darker side to them, as they attack the roots of the plants, and this causes problems in the form of large yield losses. 

"In the fight for survival, plants have developed defence mechanisms to prevent them from being attacked by the plant parasitic nematode species. They produce defences against their enemies. It is known that plants in the grass family produce benzoxazinoids or BXs, among other things, ”says Mette Vestergård Madsen.

Together with colleagues from the Department of Agroecology and Ghent University, she has used DNA analyses to investigate, how BXs affect nematodes in the soil around the plant, the rhizosphere soil - which is in the immediate vicinity of the roots - and not least inside the plant roots. 

Maize mutant enabled the experiment

Maize produces high levels of BXs, which are an important part of the plant's defences against, for example, insects. And previous studies have indicated that they also work against nematodes. But the studies have been few and only at petri dish level.

"The purpose of our study is to investigate whether the BXs have an effect on plant parasitic nematodes in maize, and in addition it could be interesting to see whether BXs have some unintended effects on other nematodes - i.e. those that are not harmful,” says Mette Vestergård Madsen.

A maize mutant that has been mutated so that the production of BXs is greatly reduced enabled the researchers' experiments. Here, a key gene has been removed from the synthesis of the BXs. And as a reference, the researchers had the mutant's mother genotype, i.e. the non-mutated variant of the same maize - also called the wild type. So, in principle, the two genotypes of maize should be similar except for the one gene, which is not found in the mutated version. 

Soil from Skælskør

Using soil from a fairly regular field in Skælskør in Denmark, the researchers made an experimental setup where the two genotypes were grown separately, but in the same type of soil from the same field.

"So, it could have been soil from any field in Denmark, because there are nematodes and plant parasitic nematodes in all soils," explains Mette Vestergård Madsen.

In the experiment, the soil was cleaned from the roots, and bulk soil, rhizosphere soil as well as roots were examined a total of four times at ten day intervals. The concentration of different BXs was measured, and not surprisingly, there were greatly reduced amounts of BX in the mutant relative to the wild type. 

DNA sequencing of nematodes

Where previously each nematode had to be found and then species-determined in a microscope, the sequencing method has made it possible to extract the DNA that is in a sample and with specific markers find the DNA that is relevant to the study. In this case, DNA from nematodes.

“So, we quickly got information about which different nematode species or families existed in the samples. In this way, we could describe the composition of the nematodes in the different samples,” says Mette Vestergård Madsen.

And the result was quite interesting. The nematode community in the soil that is not affected by plants is, according to the study, significantly different from the community found close to or in the roots. 

"In fact, we do not see much difference between the mutant and its wild-type plant. In other words, we do not see huge effects of the mutation when we look at the whole composition of the nematode community. But when we look at the roots, it becomes interesting. In the samples on day 20 and day 30 after the start of the experiment, we could see a marked difference in the nematode species called Pratylenchus neglectus,” explains Mette Vestergård Madsen. 

Pratylenchus neglectus is a plant parasitic nematode, i.e. one of the harmful species.

“We could see that it is significantly more common in the roots of the mutant than in the wild type on day 20 and day 30, so there is some evidence that when we remove these BX substances, the plant is less protected against this kind of nematodes. The same applies to some of the other plant parasitic nematode species,” says Mette Vestergård Madsen.

No adverse effect on beneficial nematodes

It is far from all species of nematodes that are negatively affected by the BXs. According to the researchers, there is just a few, but the few are in turn also some of the most harmful species. So, despite the fact that the overall composition of the nematode community varied only moderately in the BX-weakened maize mutant, the researchers found that BXs affected harmful nematodes such as Pratylenchus neglectus and Bitylenchus parvus negatively. More useful nematode species are not adversely affected, some even seemed to thrive better. In addition, the researchers were able to conclude that although some BXs also disappear into the rhizosphere, it is only the nematodes that either live on the root surface or inside the roots that are affected by the BXs. This suggests that the concentration in the rhizosphere soil is not high enough to affect the nematodes. 

"It is only the nematodes that live in close contact with the plant roots that are affected by these BXs. And according to our analyses, they only affect a few species, but they are also the most harmful of the kind, ”concludes Mette Vestergård Madsen.