Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes

Abstract

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained.Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

Does Glyphosate Affect the Human Microbiota?

“…Glyphosate targets the EPSPS enzyme by competing for the binding site with phosphoenol pyruvate (PEP) [ 20 ]. The enzyme has been categorized into four classes based on its potential sensitivity to glyphosate [ 6 , 21 ]. Classes I, II, and IV were determined based on the presence and absence of amino acid markers in active sites, whereas Class III was categorized based on a series of motifs.…”

Rethinking the Intrinsic Sensitivity of Fungi to Glyphosate

“…In bacteria and plants, the EPSPS protein sequence has a single domain, whereas fungal EPSPS proteins contain several domains [ 4 ]. Therefore, the EPSPS protein folding in fungi may result in a different interaction with the herbicide compared to the plant and bacteria EPSPS [ 8 ]. These potential effects of the multi-domain structure of the EPSPS have been mostly neglected.…”

“…These potential effects of the multi-domain structure of the EPSPS have been mostly neglected. Moreover, additional non-target mechanisms of resistance (e.g., efflux pumps, vacuolar sequestration, and metabolization of glyphosate) or sensitivity (e.g., toxic effect on the mitochondria) to glyphosate modulate the intrinsic sensitivity status in the EPSPS protein [ 8 , 27 ] and may have a differential effect on fungal species. Several experimental and field studies have shown a negative effect of glyphosate on fungal communities in soil [ 28 ] and underground host-associated interactions [ 29 ].…”

“…Glyphosate’s mode of action is competitive against the PEP and noncompetitive against the S3P [ 9 ]. However, the dual conformation of the EPSPS has been mostly studied in single-domain proteins of plants and bacteria; thus, its effect in a multi-domain structure is quite uncertain [ 8 ]. Our results showed that 354 (90.8%) fungi were potentially sensitive to glyphosate, 5 (1.3%) were resistant, and 31 (7.9%) were unknown (i.e., unclassified EPSPS proteins based on the current classification system).…”

“…Thus, glyphosate may have an effect on microbial communities of free-living and host-associated microorganisms [ 4 , 5 , 6 , 7 ]. Recent studies of the EPSPS-glyphosate relationship have provided clues on the potential effect of the herbicide on the microbiota [ 8 ]. A comprehensive analysis of the potential sensitivity of the EPSPS protein has shown the potential impact of the herbicide on several species of plant, fungi, and bacteria [ 4 , 5 ] (e.g., the herbicide has the potential to affect half of the human gut microbiota [ 4 ]).…”