Novel peptide-based agents for management of bacterial plant diseases

The findings highlight the great potential of RPMs as a crop protection agents
November 11, 2019

The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem.

 

Agricultural yields are highly damaged by plant diseases. Plant-pathogenic bacteria are among the most important causal agents of plant diseases with almost all crops being severely affected by one or more serious bacterial diseases. Chemical control of bacterial plant diseases in agriculture is highly limited. In fact, management of most bacterial plant diseases vastly relies on copper-based bactericides, which possess limited efficiency. Copper bactericides are easily washed from the plant surface by rain or overhead irrigation, conditions that, on the other hand, are highly conducive to many bacterial plant diseases.

Therefore, under these conditions, frequent sprays are needed, increasing production costs as well as environmental contamination. Remarkably, frequent sprays with relatively high concentrations of copper might cause phytotoxicity and often lead to emergence of copper resistant strains of pathogens. Importantly, copper-based products are also widely used to control plant diseases caused by fungi and oomycetes. However, the European Food Safety Authority (EFSA) has stressed that the use of copper is of concern to public health and the environment, and copper compounds are due to be gradually phased out. Considering the lack of alternatives, there is an urgent need to develop novel technologies to manage plant diseases and reduce food loss.

 

 

 

Figure 1. Synthesis of random peptide mixtures (RPMs) using a modification of the Fmoc-based solid-phase synthesis method. In each coupling step an amino acid is added from a mixture of two amino acids at equal concentrations. In the example, the amino acids are L-phenylalanine (LF) and D-lysine (DK). After 20 coupling steps, the FdK-20 RPM is generated that contains more than one million different peptides.

 

One potential alternative to the use of Cu copper bactericides are antimicrobial peptides. Antimicrobial peptides are naturally produced by eukaryotic organisms to cope with potential pathogens, being commonly named host defence peptides (HDPs). While HDPs display several antimicrobial mechanisms, many of them have been shown to disrupt bacterial membranes. Natural HDPs as well as synthetic HDP derivatives have been considered as potential agents to control pathogenic bacteria, including in agriculture. However, they might have undesired characteristics including low stability, non-specific toxicity and development of resistance by target bacteria. Importantly, the costs of production and purification of synthetic antimicrobial peptides is relatively high, thus limiting their application, particularly in agriculture.

HDPs substantially differ in their amino-acid sequence and conformation. However, most of them share a common characteristic: they are rich in both cationic and hydrophobic amino acids. Based on this knowledge, we have developed the random peptide mixture (RPM) concept. The synthesis approach is based on a modification of the conventional Fmoc-based solid-phase synthesis method, in which instead of using one pure amino acid at each coupling step, a mixture of two amino acids, in a defined proportion, is used thorough the whole synthesis process. The result is a mixture of 2n (where n is the peptide chain length) sequences of random peptides with a defined composition and controlled chain length (Figure 1). This modification significantly reduces the production cost since no purification is needed after each coupling step, as required in the conventional method.

   

Figure 2. Effects of RPMs on plant-pathogenic bacteria. (A) Minimal inhibitory concentrations (MIC) of 20-mer RPMs composed by L-phenylalanine and L- or D-lysine (FK-20 and FdK-20, respectively) on representative strains of Xanthomonas campestris pv. campestris (Xcc), Xanthomonas perforans (Xpe), Clavibacter michiganensis subsp. michiganensis (Cmm) and Pseudomonas syringae pv. tomato. All strains were highly susceptible to the RPMs except Pst that was not affected by FdK-20. The black line above the Pst/FdK bar means that no inhibition was detected at the maximal tested concentration of 200 μg/ml (modified from Topman et al., Microbial Biotechnology, 2018, 11:1027-1036). (B) Inhibition of disease symptom development in kohlrabi leaves inoculated with Xcc by protective pretreament with FdK-20 at 200 μg/ml. Left leaf, pretreated with FdK-20; right leaf, non-pretreated. Arrows indicate the sites of Xcc inoculation. 

Using the aforementioned technology, we have generated a variety of RPMs comprised of hydrophobic and cationic amino acids, with some of them having strong antimicrobial activity towards both Gram-negative and Gram-positive bacteria. Recently we showed that RPMs consisting of random 20-mer combinations of L-phenylalanine and L- or D-lysine (FK-20 and FdK-20, respectively) display powerful growth inhibition and bactericidal activities towards several phytopathogenic bacteria, including members of the Xanthomonas, Clavibacter and Pseudomonas genera (Figure 2).

Moreover, in planta studies in the greenhouse revealed that these RPMs significantly reduce disease severity of two important bacterial plant diseases: bacterial spot of tomato and black rot disease of brassicas, caused by Xanthomonas perforans and Xanthomonas campestris pv. campestris, respectively.

Importantly, in both cases, reduction of disease severity achieved by the RPMs were similar to that exerted by the commercial, copper bactericide Kocide, which was applied at 12-fold concentration of the active compound relative to the RPM treatments. Remarkably, the tested RPMs had no toxic effect on survival of honey bees and Caco-2 mammalian cells.

This study, demonstrating the potential of RPMs as novel crop protection agents, was summarized in a peer-reviewed manuscript that was published last year (Shiri Topman and colleagues, Microbial Biotechnology, 2018, 11:1027-1036).

It is important to mention that overall, 20-mer RPMs possess stronger antimicrobial activity than shorter RPMs. For example, 5-mer or 10-mer combinations of L-phenylalanine and L-/D-lysine are significantly less potent than the aforementioned FK-20 and FdK-20. Recently, we started to explore the effect of several chemical modifications in order to shorten the chain lenght of the active RPMs.

This approach could lead to further reduction of production costs, thus increasing the feasibility of the technology and making it more attractive for application in agriculture and in other fields. Importantly, we are considering several chemical modificiations that will improve the adhesive properties of RPMs and their interaction with the plant surfaces. Potentially, this could reduce washing of the compounds from the plant surface by rain or irrigation, thus providing a more efficient protection and allowing a reduction of application frequencies. In this regard, we already have some interesting chemically modified RPM candidates, which at the moment are being patented.
  
Overall, our findings highlight the great potential of RPMs as a crop protection agents. This research is being supported by a grant from the Israeli Ministry of Agriculture and Rural Development.