Crop Seeds Ready to Fight Back

Imitating Natural Self-defense Mechanisms for Pesticide Replacement

  • Fig. 1. Reaction mechanism of hydrogen cyanide formation in bitter almonds (Prunus dulcis). First, the sugar residue of the cyanide containing glycoside amygdalin is cleaved with the help of the enzyme β-glucosidase resulting in mandelonitrile and two molecules of glucose. Second, mandelonitrile is degraded to benzaldehyde and hydrogen cyanide. The second step can be catalyzed by the enzyme hydroxynitrilelyase (HNL), but is proceeding also fast enough without enzyme catalysis. Figure adapted from [1].Fig. 1. Reaction mechanism of hydrogen cyanide formation in bitter almonds (Prunus dulcis). First, the sugar residue of the cyanide containing glycoside amygdalin is cleaved with the help of the enzyme β-glucosidase resulting in mandelonitrile and two molecules of glucose. Second, mandelonitrile is degraded to benzaldehyde and hydrogen cyanide. The second step can be catalyzed by the enzyme hydroxynitrilelyase (HNL), but is proceeding also fast enough without enzyme catalysis. Figure adapted from [1].
  • Fig. 1. Reaction mechanism of hydrogen cyanide formation in bitter almonds (Prunus dulcis). First, the sugar residue of the cyanide containing glycoside amygdalin is cleaved with the help of the enzyme β-glucosidase resulting in mandelonitrile and two molecules of glucose. Second, mandelonitrile is degraded to benzaldehyde and hydrogen cyanide. The second step can be catalyzed by the enzyme hydroxynitrilelyase (HNL), but is proceeding also fast enough without enzyme catalysis. Figure adapted from [1].
  • Fig. 2. Build-up and mode of action of the cyanogenic coating as defense mechanism for wheat grains. Alternating layers of enzymes (pink), polylactic acid (PLA, blue), and in PLA embedded amygdalin (orange) are applied on uncoated wheat grains. The transparent coating has a thickness of 30-50 µm (scale bar: 50 µm). If feeding insects (here shown: larvae of the meal beetle, Tenebrio molitor) damage the coating, the released substances react with each other, e.g. the enzyme β-glucosidase converts amygdalin into hydrogen cyanide. This can weaken or kill the insect at high doses. Figure adapted from [1].
By applying biomimetics, the scientific term for imitating biological systems or strategies, already numerous complex problems of humankind could be solved in an innovative manner. In this article it is shown how a defense strategy of plants belonging to the genus Prunus could be copied in such a way that it became possible to use it as protection for crop grains during storage against a variety of insect pests without employing conventional pesticides. When the pests tried to feed on the grains, chemicals and enzymes mixed and small amounts of gaseous hydrogen cyanide were released which is toxic and repellent for insects.
 
After millions of years of adaptation to their environment and co-evolution with  natural predators, many bio-organisms have evolved effective defense strategies. Some of these strategies, e.g. antibiotics produced by molds, have been successfully applied in medicine. Plants have also developed an abundant number of different mechanisms that protect them from herbivores, such as insects, or pathogens ,such as bacteria and fungi. For example, many species of the plant genus Prunus, such as apricots, bitter almonds or peaches, protect their seeds with hydrogen cyanide (HCN). If herbivores try to open the seeds, cyanide-containing precursor molecules and specific enzymes are mixed leading to the release of the gaseous natural cell toxin hydrogen cyanide that is able to repel or even kill insects and other herbivores (fig. 1). This kind of defense has inspired us to develop a biomimetic material that performs and functions similarly compared to its natural counterpart, but is flexibly applicable, e.g. as coating for crop seeds. The advantages of this self-defending material are its ability to react only when triggered by attacks and that all material components are completely biodegradable.
 
Hydrogen Cyanide Protection for Seeds
 
Recently, we could show in two scientific studies, that the cyanogenic material is very well suited as coating for wheat grains to protect them from insect pests during storage, which is still a significant problem worldwide.

The germination of seeds after storage was negligibly influenced by the coating, which is an important precondition for their applicability [1, 2].

 
The biomimicked coating has a thickness of 30-50 µm and consists of different layers of polylactic acid (PLA), a biodegradable polymer, which stably separates the different compartments harboring either enzyme (β-glucosidase) or cyanide precursor molecules (amygdalin). Amygdalin is the same cyanogenic glycoside found in the shell of bitter almond seeds (Prunus dulcis). To determine the most efficacious layer composition and order, different layer sequences were tested. In general, the cyanogenic potential, i.e. the amount of released hydrogen cyanide, increased proportionally with the number of amygdalin-containing layers. Additionally, previous experiments had already shown that the enzymes have to be located in the most inner layer of the coating to be most effective. On top of this lies a layer of pure polylactic acid, followed by one to two layers in which the hydrogen cyanide precursor amygdalin is embedded – the same substance found in the husks of bitter almond seeds. A final layer is composed of pure PLA.
 
If an insect chews through these layers, the amygdalin is released, followed by the enzyme. The two substances mix together and the amygdalin breaks down into hydrogen cyanide, which is a deterrent for insects and inhibits their metabolism (fig. 2).
 
Successful Testing on Insect Pests
 
In collaboration with the Julius-Kühn Institute in Berlin, the efficacy of the cyanogenic coating on a variety of cereal pests was tested. The biomimicked defense system proved very effective against the larvae of the mealworm (Tenebrio molitor), the Indian mealmoth (Plodia interpunctella) and the lesser grain borer (Rhizopertha dominica). The lesser grain borer is a beetle that causes considerable damage to wheat stores worldwide.
 
Significantly fewer fully grown beetles hatched on coated than uncoated seeds. They reproduced less successfully and the larvae grew more slowly because they had eaten less.
 
However, the layering didn’t keep absolutely all insects from feasting on the wheat grains: the treatment was not effective against the wheat weevil (Sitophilus granarius). This type of beetle does not lay its eggs on the grain, but instead bores a hole into it to deposit the eggs and seals it up afterwards. The larvae then eat the wheat grain from the inside out, which means that they don’t come into contact with the here presented, chemically active coating.
 
Field Trials Confirm Applicability
 
In laboratory and fieldwork, it was demonstrated that the coating did not impair the germination of wheat grains. In the lab, 98% of the coated grains germinated. In the field, the coated grains did germinate a little later than the uncoated ones, and the seedlings initially developed more slowly. Nonetheless, the wheat plants were able to recover this initial deficit during later growth stages.
 
Outlook
 
Overall, we were able to show that a completely biodegradable material imitated from apricots and peaches can be used as coating for wheat grains endowing them with additional defense capabilities against insect pests. The additional cost for the coating are expected to be around 6-10 % per kilogram of wheat grains. In future studies, it has to be investigated, whether the cyanogenic coating against insect pests can be also applied to other crop seeds, such as corn or rice, or other things worth protecting. Nevertheless, the biomimetic coating has the potential to serve as alternative for conventionally used grain pesticides.

Authors
Carlos Mora1 and Wendelin Stark1

 
Affiliation
ETH Zurich, Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, Zurich, Switzerland
 
Contact
Dr. Carlos Mora
ETH Zurich
Department of Chemistry and Applied Biosciences
Institute for Chemical and Bioengineering
Functional Materials Laboratory
Zurich, Switzerland
carlos.mora@chem.ethz.ch
 

References
[1] C. A. Mora, J. G. Halter, C. Adler, A. Hund, H. Anders, K. Yu, W. J. Stark, Application of the Prunus spp. cyanide seed sefense system onto wheat: reduced insect feeding and field growth tests, J. Agr. Food Chem. 64, 3501–3507(2016) – DOI: 10.1021/acs.jafc.6b00438

[2] J. G. Halter, W. D. Chen, N. Hild, C. A. Mora, P. R. Stoessel, F. M. Koehler, R. N. Grass, W. J. Stark, Induced cyanogenesis from hydroxynitrile lyase and mandelonitrile on wheat with polylactic acid multilayer-coating produces self-defending seeds, J. Mater. Chem. A 2, 853-858 (2014) – DOI: 10.1039/C3TA14249C

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