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.

Purpose In cold climates, glyphosate residues may linger in soils, with effects on plant–microbe interactions and, consequently, plant performance. Here, we explore the influence of glyphosate residues on the endophytic microbiota (bacteria and fungi) and performance of the perennial nitrogen-fixing weed Lupinus polyphyllus. Methods In a common garden, we grew plants from six populations of L. polyphyllus in glyphosate-treated or untreated control soils, with or without additional phosphorus. We sampled plant microbiota (leaves, roots, nodules) and assessed plant performance based on six traits: height, retrogression probability (i.e. shrinkage), biomass, root:shoot ratio, nodule number, and nodule viability. Results The richness of plant endophytic microbial communities was determined by soil phosphorus level rather than by glyphosate treatment. However, for bacteria, the composition of these communities differed between glyphosate-treated and control soils across plant tissue types; no difference was observed for fungi. The plant bacterial communities in both soil types were dominated by potential nitrogen-fixing bacteria belonging to family Bradyrhizobiaceae, and particularly so in glyphosate-treated soils. Overall, though, these changes in plant bacterial communities had a minor effect on plant performance: the only difference we detected was that the probability of retrogression was occasionally higher in glyphosate-treated soils than in control soils. Conclusion Our findings indicate that glyphosate-based herbicides, when applied at the recommended frequency and concentration, may not have critical effects on the growth of short-lived weeds after the safety period has passed; however, the endophytic microbiota of such weeds may experience longer-lasting shifts in community structure.

Plants harbor a large diversity of endophytic microbes. Meadow fescue (Festuca pratensis) is a cool-season grass known for its symbiotic relationship with the systemic and vertically—via seeds—transmitted fungal endophyte Epichloë uncinata, yet its effects on plant hormones and the microbial community is largely unexplored. Here, we sequenced the endophytic bacterial and fungal communities in the leaves and roots, analyzing phytohormone concentrations and plant performance parameters in Epichloë-symbiotic (E+) and Epichloë-free (E-) individuals of two meadow fescue cultivars. The endophytic microbial community differed between leaf and root tissues independent of Epichloë symbiosis, while the fungal community was different in the leaves of Epichloë-symbiotic and Epichloë-free plants in both cultivars. At the same time, Epichloë symbiosis decreased salicylic acid and increased auxin concentrations in leaves. Epichloë-symbiotic plants showed higher biomass and higher seed mass at the end of the season. Our results demonstrate that Epichloë symbiosis alters the leaf fungal microbiota, which coincides with changes in phytohormone concentrations, indicating that Epichloë endophytes affect both plant immune responses and other fungal endophytes. Whether the effect of Epichloë endophytes on other fungal endophytes is connected to changes in phytohormone concentrations remains to be elucidated.

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.

Plants harbor a large diversity of endophytic microbes. Meadow fescue (Festuca pratensis) is a cool-season grass known for its symbiotic relationship to the systemic and vertically- via seeds - transmitted fungal endophyte Epichloë uncinata but the effect of the endophyte on the microbial endophyte community and phytohormones is largely unexplored. Here, we sequenced the endophytic bacterial and fungal communities in the leaves and roots, analyzed phytohormone concentrations and plant performance parameters in Epichloë-symbiotic (E+) and Epichloë-free (E-) individuals of two meadow fescue cultivars. The endophytic leaf microbial community differed between leaf and root tissues independent of endophyte symbiosis while the fungal community was different in leaves of Epichloë-symbiotic and Epichloë-free plants in both cultivars. At the same time, endophyte symbiosis decreased salicylic acid and increased auxin concentrations in leaves. Epichloë-symbiotic plants showed a higher biomass, chlorophyll content (SPAD) and higher seed mass at the end of the season. Our results demonstrate that Epichloë-symbiosis alters the leaf fungal microbiome, which coincides with changes in phytohormone concentrations, indicating that Epichloë endophytes affect both, plant immune responses and other fungal endophytes. Whether the effect of Epichloë endophytes on other fungal endophytes is connected to changes in the phytohormone concentrations remains to be elucidated.