If you plan to collect walnuts in the fall you would be well advised to wear waterproof gloves, since, like the leaves and other parts of the plant, the green shells contain hydrojuglone. When hydrojuglone is exposed to air, it forms juglone – a substance that turns our skin black. On the other hand, it can also kill fungi and plants. “That's why you find hardly any other plants growing under walnut trees,” explains Judith Wirth, who heads the “Herbology in Field Crops” research group at the Agroscope agricultural research center in Switzerland. There, researchers looking for alternatives to conventional weed management (by herbicide) are increasingly becoming aware of the growth-inhibiting effect of certain plants. In technical language this phenomenon is known as allelopathy.
“There are many theories about how allelopathy develops. But there are only isolated instances where we have real evidence of the substances exuded by plants and the way in which this occurs. Walnuts are one such example,” says Wirth. The hypothesis is that certain plants used as cover crops achieve this effect via root exudates. For her investigations, Wirth joined forces with experts from the University of Natural Resources and Applied Life Sciences (BOKU) in Vienna in the project “Crop-weed interaction in the soil”. One of these experts is soil ecologist Markus Puschenreiter from the Institute of Soil Research, a specialist in root-soil interaction. The analytical-chemical input comes from the team of Stephan Hann, head of the Institute of Analytical Chemistry at BOKU.
The root region
A living root influences the soil surrounding it. “This is where a wide variety of processes take place,” explains Puschenreiter. “The roots release substances called exudates in order to mobilize nutrients, change soil properties or trigger symbiotic relationships. In addition, via the root the plant interacts with surrounding organisms, be they microorganisms or other plants.” In the context of the project, the researchers are interested in specific chemical signals that have a negative effect on the development of neighboring plants.
Since it is difficult to examine the soil portion directly surrounding the root, the team designed a first intermediate stage for their experiments: the plants they want to investigate are grown not in soil but in glass bead cultures, and they are supplied with a nutrient solution. Root exudates are collected in this solution and can thus be secured for analysis. “Of course this is a highly simplified set-up compared to natural soil,” Puschenreiter concedes, “but previous projects have taught us that it is a necessary step to be able to detect the growth-suppressing substances in the first place.” For the trial, the team cultivates common buckwheat (Fagopyrum esculentum) along with lopsided oat (Avena strigosa) as a second crop. As a test weed, the researchers are using amaranth (Amaranthus retroflexus). “We based our selection on available experience. Both crops have long been used as cover crops and are known to suppress weeds well,” says Wirth.
New cultivation method
To achieve optimal conditions, other processes also had to be designed specifically for the trial. “First of all, the plants have to grow in a reproducible and controlled manner, which is not easy to do. And sampling is also a challenge,” says Hann, who is in charge of the chemical analyses. On the one hand, it must be ensured that the substances do not permute on their way from the root to the lab.
On the other hand, the team was also concerned with finding a valid control method. This is why they developed a novel cultivation method in the course of the project, in which the root of one plant grows in two different containers (“split-root”). In this way, the root exudate can be compared with the control exudate from a second container in which the same plant is growing without the presence of any weeds or other plants. In addition to identifying molecules with an allelopathic effect, the researchers also want to find out whether the composition of the exudates changes depending on the presence of a weed or other plants.
From analysis to application
At BOKU, the root exudates that end up in the nutrient solutions of the various plant combinations are analyzed for their composition. Stephan Hann's team uses mass spectrometry to do that – an analytical method that can determine the mass of a molecule very accurately. “This method detects about 1,000 different substances in any given one of our samples,” says Hann. Many of these finds are general metabolic products of plants or microorganisms and thus not relevant to the issue under study. It takes statistical analysis to reveal which substances are excreted only in the presence of a weed. According to Hann, this is where the real analytical challenge begins. “ Mass spectrometry only provides a signal from which we can compute the molecular formulas in question, i.e. which elements are present in the still unknown substances and in what numbers. But each molecular formula can hide hundreds of chemical structures to which the individual atoms can be attributed. Hence, the analysis never tells us exactly which substance is present.”
First results of improved analysis
According to Hann, this problem has been known for a long time and arises in many areas where mass spectrometry is used. “Among other things, it complicates the identification of biomarkers in medicine or analyses from the environmental field, when substances that have been inadvertently discharged into bodies of water need to be identified.” Part of the project's research output, he said, is therefore to develop new ways for approaching this non-targeted analysis. That involves first of all the generation of structure-specific fragments that can be used for identification via special algorithms and database matching. The project group recently presented initial results at the annual conference of the American Society for Mass Spectrometry – the world's largest conference on mass spectrometry – in Houston.
As soon as the methodological and analytical obstacles have been mastered, the team intends to extend the approach to experiments in soil instead of in glass bead culture. Only then can the actual situation in the field be investigated. The researchers expect that interaction will be more complex in soil. “The inhibitory effect of a plant is always the result of an interaction of many factors. Some plants are simply more competitive and better at taking up water or nutrients. Allelopathy is only part of the inhibitory effect and likely varies as a factor of aspects such as plant stress, water status, nutrient availability, life cycle and so on,” notes Wirth. “Ideally, we will be able to identify individual allelopathic metabolites.” Armed with this knowledge, Wirth explains, one can breed one's own varieties that exude these substances in greater quantities. However, even obtaining more basic knowledge about the conditions of inhibitory plant interactions already constitutes an asset for agriculture, enabling farmers to establish sustainable methods.
Stephan Hann, an expert in chromatography and mass spectrometry, has been heading the Institute of Analytical Chemistry at the University of Natural Resources and Life Sciences, Vienna (BOKU) since 2020. Markus Puschenreiter also holds a position at BOKU, at the Institute for Soil Research. Among other things, Puschenreiter does research on the topic of rhizosphere ecology.
Judith Wirth specializes in weed science. She heads the “Herbology in Field Crops” research group at Agroscope in Switzerland, a center of excellence for agricultural research of the Swiss Federal Office for Agriculture. The bilateral, interdisciplinary project “Crop-weed interaction in the soil” was set to run for three years until the end of 2023. It received roughly EUR 175,000 in funding from the Austrian Science Fund FWF.
Publications and contributions
Bennett A., Eroğlu C., Steininger‐Mairinger T., Puschenreiter M., Gfeller A., Wirth J., Hann S.: Dual column chromatography improves non‐targeted analysis coverage when assessing rhizosphere chemical communication, in: Book of Abstracts of the 71st ASMS Conference on Mass Spectrometry and Allied Topics, June 04-08, 2023, Houston, Texas, USA
Gfeller A., Glauser G., Etter C., Signarbieux C., Wirth J.: Fagopyrum esculentum Alters Its Root Exudation after Amaranthus retroflexus Recognition and Suppresses Weed Growth, in: Frontiers in Plant Science 9, 2018