Previous Article in Journal
Using Moss Walls for Air Quality Monitoring: Extending Their Utility Beyond Traditional Green Infrastructure
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJPB
Volume 16
Issue 2
10.3390/ijpb16020045
ijpb-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Altered Translocation Pattern as Potential Glyphosate Resistance Mechanism in Blackgrass (Alopecurus myosuroides) Populations from Lower Saxony

by
Markus Radziewicz
1,
Dirk M. Wolber
1,
Thomas Pütz
2 and
Diana Hofmann
2,*
1
Chamber of Agriculture Lower Saxony, Plant Protection Office, 30453 Hannover, Germany
2
Forschungszentrum Jülich GmbH, Institute for Bio- and Geosciences, IBG-3: Agrosphere, 52428 Jülich, Germany
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2025, 16(2), 45; https://doi.org/10.3390/ijpb16020045
Submission received: 16 July 2024 / Revised: 10 March 2025 / Accepted: 25 March 2025 / Published: 16 April 2025
(This article belongs to the Section Plant Response to Stresses)
Browse Figure
Versions Notes

Abstract

:
Glyphosate is a broad-spectrum herbicide widely used. After years of extensive usage, many weed species have developed resistance due to both target-site (TSR) and non-target-site resistance mechanisms (NTSRs). Alopecurus myosuroides is a competitive weed species. Greenhouse monitoring trials in Germany have revealed reduced glyphosate efficacy against some populations of Alopecurus myosuroides. In a foregoing dose–response study, individual plants from four out of six tested populations survived full (1800 g a.i. ha−1) or double (3600 g a.i. ha−1) glyphosate dose rates permitted, suggesting the presence of tolerant biotypes with yet unknown resistance mechanisms. Our aim was to investigate the absorption and translocation patterns of glyphosate in these biotypes. The plants were first treated with 14C-glyphosate, and 14C-glyphosate absorption and translocation were subsequently visualized by phosphorimaging and finally quantified by liquid scintillation counting. The results showed significant differences in the distribution of glyphosate in different plant organs, with significantly more being translocated out of the treated leaf in glyphosate-resistant compared to sensitive (S-) biotypes. The study’s findings are partly in contrast to previous studies that have found reduced translocation. Our study demonstrates the complex nature of glyphosate resistance and suggests further experiments to finally elucidate the underlying resistance mechanisms in the biotypes of the Alopecurus myosuroides studied.

1. Introduction

Glyphosate is a highly effective, non-selective broad-spectrum, systemically active herbicide [1]. It has become the most widely used herbicide worldwide since its introduction into the market in 1974 [1,2]. It is used throughout the world in crop areas for controlling weeds, preparing seed beds, desiccating cover crops, and facilitating harvests [3,4]. Furthermore, glyphosate is used worldwide in tree, nut, and vine cultivation besides various non-crop areas like railway grounds for weed control [1,3,5]. Since the creation and subsequent introduction of glyphosate-resistant crops into agriculture in various countries, glyphosate has been widely used in these regions as a selective in-crop herbicide for weed control [1,3,6,7].
After application and subsequent absorption into plants, glyphosate is translocated to the meristematic tissues via phloem transport [8,9], where it finally passes through the plastid membrane into the plastids of cells [8]. There, it inhibits the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS). This translocation is essential for the efficacy of the herbicide [8,9], as the highest contents of EPSPS are found in plant meristems [9].
Glyphosate highly effectively inhibits EPSPS in the shikimate pathway. Normally, the EPSPS binds phosphoenolpyruvate (PEP) and combines it with shikimate-3-phosphate (S3P) to form 5-enolpyruvyl-shikimate-3-phosphate (EPSP). Due to the similar structure to PEP, glyphosate binds to the EPSPS and thus blocks the binding site, which is no longer available for PEP. As a result, the biosynthesis of the aromatic amino acids Tyr, Phe, and Trp in the shikimate pathway was inhibited and instead the intermediate shikimate accumulates in the treated plants [1,4,5]. The resulting metabolic disturbances cause first growth inhibition (hours to days after glyphosate application), followed by the yellowing of leaves several days later [10], before leading finally to plant death [5].
Following years of extensive glyphosate usage, the first naturally evolved resistance to glyphosate has been reported in a population of rigid ryegrass (Lolium rigidum Gaud.) in Australia [11]. Since then, the number of weed species developing resistance to glyphosate has steadily increased. Today, resistant populations of numerous different weed species according to the criteria defined by the Herbicide Resistance Action Committee (HRAC) [12] were documented [13].
As mechanisms, both target-site resistance (TSR) as well as non-target-site resistance (NTSR) to glyphosate were discovered so far. TSR mechanisms include mutations in the EPSPS gene, leading to amino acid substitutions in the EPSPS. In most of the reported cases linked to this mechanism, proline at position 106 in the EPSPS molecule is substituted by another amino acid (e.g., Ser, Ala, Thr, or Leu), which leads to a structural change in the glyphosate binding site. This decreases the affinity between the EPSPS and glyphosate [5,14,15]. Furthermore, additional mutations together with the Pro-106 mutation [16,17,18,19] or alone as a single different mutation (Thr-102-Ser) are known today [20].
Another TSR mechanism is an increased EPSPS gene duplication/amplification. In this case, the resistant plant contains more copies of the EPSPS gene compared with a susceptible plant, leading to an increased synthesis/higher expression of the EPSPS. As a result, higher concentrations of EPSPS are present than the recommended doses of glyphosate [5,14,15,21]. Today, two distinct mechanisms are known, which lead to this type of TSR: I) a tandem duplication mechanism and II) a large extrachromosomal circular DNA (eccDNA) that is tethered to the chromosomes and passed to gametes at meiosis [21].
Known NTSR resistance mechanisms include a lower foliar retention of glyphosate [22] and reduced glyphosate absorption through the leaf surfaces [22,23,24,25,26]. Differences in the leaf cuticle can contribute to the lower retention and absorption of the glyphosate spray solution [27]. Besides the amount or thickness of the cuticular wax mass [28] or their chemical properties [27], presumably, differences in the morphology of the leaf surface itself, such as different numbers of trichomes (hair-like structures with different functions), can also lead to lower absorption [28].
Another NTSR mechanism is the alteration of glyphosate translocation in the plant; most of the absorbed glyphosate is retained in the treated leaves, and consequently, less is translocated to the rest of the plant [29,30,31,32]. This mechanism correlates in many cases with the rapid sequestration of glyphosate into the vacuole of plant cells so that less glyphosate is available for translocation. Behind this vacuolar sequestration, an active transport process through the tonoplast is assumed, in which one or more tonoplast transporters—probably ABC-transporters—are involved [8,14,33].
Further NTSR mechanisms discovered to date include the following: (I) enhanced metabolic degradation of glyphosate through an increased synthesis of the enzyme aldo-keto reductase, which degrades glyphosate to aminomethylphosphonic acid and glyoxylate [34], (II) rapid necrosis of glyphosate-treated mature leaf tissue, resulting in the reduced translocation of glyphosate to meristems which continue growth [35,36], and (III) extrusion of glyphosate from the cytoplasm into the extracellular space, likely by an up-regulated plasma membrane-localized transporter [37].
In addition, there is evidence of still unknown resistance mechanisms [35,36,38,39,40,41,42,43,44]. All glyphosate resistance mechanisms can occur alone or in combination within populations or individuals [14,44].
Glyphosate-resistant weed populations have not been a problem in Germany so far. However, two recent studies from Rhineland-Palatinate/Germany show that (I) a population of the weed species perennial ryegrass [Lolium perenne (L.)] can no longer be controlled with the recommended glyphosate field rate in Germany [45] and (II) a possible resistance development to glyphosate occurred in a population of annual fescue [Vulpia myuros (L.)], respectively [46,47].
Since 2015, regularly conducted greenhouse monitoring trials on herbicide resistance occurrence and their potential spread in Lower Saxony/Germany revealed a reduced glyphosate efficacy against some populations of Blackgrass [Alopecurus myosuroides (Huds.)]. Alopecurus myosuroides is probably the most important herbicide-resistant weed in Germany [48] and across Europe [49,50]. It is a competitive weed species that can cause high yield losses if not adequately controlled [49]. Individual plants in recent bioassays occasionally survived the recommended field rate of 1800 g glyphosate ha−1 (g a.i. ha−1) or even higher rates. Among survived plants, neither Pro-106 mutations in the EPSPS gene nor EPSPS gene duplication were detected [51]. In various countries, mainly in Europe, to date Alopecurus myosuroides evolved resistance to herbicides with six modes of action [13], according to the criteria established by HRAC, among them also against glyphosate with the mode of action of EPSPS inhibition HRAC Group 9.
In 2019, the authors conducted a dose–response study with six Alopecurus myosuroides populations from Lower Saxony, for which a reduced glyphosate sensitivity is suspected [52]. No statistically significant reduction in sensitivity in any of the tested populations compared to a sensitive (S) reference population could be detected. However, some individual plants from four out of six tested populations survived the full (1800 g a.i. ha−1) or double (3600 g a.i. ha−1) glyphosate dose rate permitted in Germany without any visible damage and continued to grow vitally. These survivors are assumed as tolerant individuals with an unknown resistance mechanism. The aim of this study was to investigate the absorption and translocation patterns of glyphosate in these selected individuals to confirm or disprove these possible, known NTSR mechanisms. Due to the controversial nature of the topic, an independent public institution was entrusted with the investigation.
These observed survivors are explicitly not plants that grew after glyphosate application—the emergence of additional plants in the pots of the dose–response study was regularly controlled, and newly emerging plants were removed. The survivors examined in the present study must therefore be postulated as tolerant biotypes with a yet unknown resistance mechanism. As these are therefore only (initial) individual plants (further called individuals), no plant material was or is currently available for further scientific investigations. However, we believe that the economic relevance and scope of these observations require early publication, as follows:
(I) Should Alopecurus myosuroides develop widespread tolerance to this broad-spectrum herbicide (including in Germany), this would have massive economic consequences—as a further challenge among the challenges in agriculture, such as climate change.
(II) The scientific understanding of the mechanism (s) is the first step and thus the basis for all subsequent reactions and decisions—from changing application recommendations to any necessary substitution of the herbicidal active ingredient. In the event of the next indications of glyphosate resistance development in weed populations, the present study should help to classify this and to mitigate or prevent it through suitable measures in crop production.
For highly sensitive and spatially resolved characterization, radioactive 14C-glyphosate was applied. First, “his translocation” was tracked non-destructively using phosphorimaging, and subsequently quantified by liquid scintillation counting (LSC) after the combustion with an oxidizer of all before separated plant compartments.

2. Materials and Methods

2.1. Plant Material

Plants that survived the dose–response study mentioned above at 1800 (P11, P39, and A1.3) and 3600 g a.i. ha−1 (A1.2 and A2.2), as well as plants of a sensitive reference population each without (S-0) and with previous treatment at 225 g a.i. ha−1 (S-0125), were vegetatively propagated (cloned) to obtain enough identical plant material for the subsequent studies (Table 1). To differentiate between the variants of S, the population ID was extended by an additional number (e.g., S-0125 for 0.125 × full glyphosate dose rate permitted in Germany).

2.2. Propagation and Growing Conditions

First, plastic foil (low density polyethylene) and hereon irrigation fleece were laid out in plant trays. Plastic pots (polypropylene, 9 cm diameter) were filled with a mixture of three parts soil (strong loamy sand, pH 6.8, organic matter 1.9%, sterilized by steaming at 100 °C for 30 min) and one part perlite (pH 7, air pore volume 95%), subsequently placed in the plant trays.
When the selected precursor plants reached the 8 to 9 tillers stage (BBCH 28-29), they were carefully removed from the pots (Jiffypot®, round, 250 mL volume, Jiffy Products International BV, Lindtsedijk 20a, Zwijndrecht, The Netherlands) in which they were previously cultivated and separated from soil by washing their roots under water. The tillers of each plant were then separated with a razor blade. All except two or three leaves on each tiller were cut off. Roots as well as the remaining leaves were cut back, and the separated tillers were planted one per pot in the prepared plastic pots. The plants developed in a greenhouse (mid-April to mid-May). The moisture level was checked daily and subirrigation was conducted as needed. Twice (21 and 30 days after propagation), a nutrient solution Hakaphos® Red ((8% N, 12% P2O5, 24% K2O, 4% MgO, 0.01% B, 0.02% Cu, 0.075% Fe, 0.05% Mn, 0.001% Mo, 0.15% Zn; 1 g/L), COMPO EXPERT GmbH, Krögerweg 10, Münster, Germany) was added.

2.3. 14C-glyphosate Treatment

As (radiolabeled) treatment solution, 14C-glyphosate ([glycine-2-14C], 99 % radiochemical purity, declared by calibration certificate 1.85 MBq in 500 µL water, American Radiolabeled Chemicals, 101 Arc Drive, Saint Louis, MO 63146, USA), together with 9.375 µL of a non-labeled, commercially available potassium salt formulation of glyphosate (Roundup® PowerFlex, 480 g a.i./l, MONSANTO Europe S.A./N.V, Haven 627 Scheldelaan 460, Antwerp, Belgium) and 300.625 µL distilled water, was prepared to reach the final concentration equivalent of 2898 g a.i. ha−1 in 200 L of water, which corresponds to the 1.61 x full glyphosate dose rate permitted in Germany.
Aliquots of this solution, containing 21.7 kBq each, were applied for the treatment of one selected leaf each per plant. Two to three clones each of the surviving plants were used (Table 1). After plants reached their 7 tillers to 1 node stage (BBCH 27-31, Table 1), the first one of the younger, fully expanded leaves of each plant was completely wrapped in aluminum foil. Subsequently, each plant was sprayed with non-radioactive glyphosate at 1800 g a.i. ha−1 using a fine sprayer (Hobby 05 Flex, GLORIA Haus- & Gartengeräte GmbH, Därmannsbusch 7, Witten, Germany) in a spiral sprayer movement from the plant center to outside. Thereby, three to four manual spray shots were applied depending on the individual plant size. After this first, cold herbicide treatment, sprayed plants were left for 30 min in a fume hood to dry. Subsequently, aluminum foils were removed. The second application onto the previously wrapped leaves took place with 10 µL of the above blended 14C-glyphosate treatment solution, applied with a Hamilton syringe as 20-30 finest droplets to the adaxial surface. This special, different approach is necessary for reasons of radiation protection (prevention of inhalation during spraying) and for precise dosing of the labeled herbicide. In addition, an even application of the pure chemical on the wax surface of plants is not possible directly, only as a surfactant-containing formylation.

2.4. Harvest and Sample Preparation

Plants were harvested 240 and 264 h after treatment, respectively (HAT, Table 2). First, the treated leaf (TL) of each plant was removed at the ligula. The leaf blade was defined as a leaf, the leaf sheath as belonging to the shoot (pseudostem). The shoots of some grasses, such as Alopecurus myosuroides, consist of many nested leaf sheaths. The TL was immersed in a polypropylene tube filled with 10 mL distilled water and alternately 20-25 s vortexed, gently shaken and swung to remove adsorbed, i.e., non-absorbed 14C-glyphosate from the leaf surface. This leaf wash solution was mixed with 10 mL scintillation cocktail (Ultima Gold XR, PerkinElmer Inc., 940 Winter St, Waltham, MA, USA), then measured using a liquid scintillation counter (LSC, Tri-Carb 3110 TR, PerkinElmer Inc., 940 Winter St, Waltham, MA, USA), freshly calibrated daily in the single dpm mode, with background correction by the IPA standard. The TL was then wrapped in a paper towel to dry.
The roots of each plant were carefully washed free of soil using successive water baths, and adherent perlite was mechanically removed by hand. Aliquots of the subsequently filtered root wash solutions were taken, mixed with 10 mL of scintillation cocktail and measured by LSC, too.
Above-ground plant organs were divided from the roots (ROs). The roots were blotted dry and wrapped in paper towels. Similarly, the (complete or subdivided) above-ground plant organs were evenly spread for later imaging between paper layers. All such prepared plant samples were oven dried at 60 °C for 168 h in a horizontal position.

2.5. Phosphorimaging

Phosphorimaging was used to obtain an overall picture of the plants and to visualize the 14C-glyphosate distribution in the plant. Our previously optimized, analogous to [54] two-step erasing process was conducted in order to reset the storage capacity and to reach a low blank of the phosphor imaging plates used (DÜRR NDT CR 35, 20 × 40 cm and 35 × 50 cm, DÜRR NDT GmbH & Co. KG, Höpfigheimer Straße 22, Bietigheim-Bissingen, Germany; Fujifilm BAS-MS 2040, 20 × 40 cm, FUJiFILM, Akasaka, Minato City, Tokyo, Japan), as follows: the imaging plates were first erased for 30 min under a high-energy white light (Erasing Unit of a Bio-Imager FUJI BAS 1000, FUJiFILM, Akasaka, Minato City, Tokyo, Japan) and immediately before starting exposure with the Bioimager CR35 Bio (Elysia-Raytest GmbH, Benzstraße 4, Straubenhardt, Germany).
To protect the plates against humidity, dust, and any contaminations, they were wrapped first into very thin cellophane foils. All parts of each plant were placed together onto the so protected imaging plate (s), evenly spread, covered successively by paper, soft foam, and an acrylic glass on the top to optimize the contact between plant parts and the imaging plate. The exposure time (in darkness) was previously optimized to 42 h. After exposure, all coverings including the plants themselves were removed, and the imaging plates were—still in the dark—immediately scanned with the Bioimager CR 35 Bio (before optimized: sensitive mode, 100 µm resolution), subsequently analyzed with the data analysis program Aida Image Analyzer v.5.0 and presented as rainbow color images (using the same optimized gain factor), exported to power point for publication (Elysia-Raytest GmbH, Benzstraße 4, Straubenhardt, Germany). The panel quality is routinely tested using a series of commercially available 14C polymer references (ELYSIA-Raytest, Straubenhardt, Germany).

2.6. Absorption and Translocation

After phosphorimaging, above-ground plant organs were further divided in the untreated leaves (ULs) and the pseudostem region (PS), respectively. All plant parts of each plant were weighed out, divided into subsamples, and combusted in a biological oxidizer (Hidex Oxidizer 600 OX, Hidex, Lemminkäisenkatu 62, Turku, Finland). Evolved 14CO2 was immediately trapped in OxySolve C-400 cocktail (Zinsser Analytics, now Gardner Denver Thomas GmbH, Fürstenfeldbruck, Germany) and subsequently measured by LSC as well. All values were corrected for the daily parallel estimated instrument recovery rate (consistently proven over 95%.).
The proportion of 14C-glyphosate absorbed/translocated was calculated with the following equation:
(TL + UL + PS + RO)/[(TL + UL + PS + RO) + leaf wash solution] × 100
whereby (TL + UL + PS + RO) represents the sum of radioactivity in the plant parts. The 14C-glyphosate in the root wash solutions was determined as several aliquots, and a calculated mean value was used for further calculations.

2.7. Statistical Analysis

Analysis of variance (one-way ANOVA) was conducted using GraphPad Prism 8.4.3 (GraphPad Software, 2365 Northside Dr. Suite 560, San Diego, CA 92108, USA) to test for significant differences between biotypes in 14C-glyphosate absorption, translocation, and recovery. The model assumption of normal data distribution was inspected graphically (QQ-Plot) and with several normality tests (D’Agostino–Pearson omnibus K2 test, Anderson–Darling test, Shapiro–Wilk normality test). The model assumption of homogeneity of variance was inspected graphically (homoscedasticity plot) and with a Brown–Forsythe test. The data for absorption, translocation, and radioactivity levels within the treated leaf, untreated leaves, and the pseudostem were normally distributed (p > 0.05).
In contrast, the data for radioactivity levels within the roots and recovery of radioactivity were not normally distributed and therefore transformed (arcsine of the square root) prior to ANOVA to meet model assumptions. Means were separated using Fisher’s LSD test at a 5% level of probability (p = 0.05).

3. Results

The 14C-recovery in our study was on average 85.8% of applied radioactivity (aR) for all biotypes investigated. The remarkable difference of 14.2% to the originally applied 14C may have two main reasons, as follows: (I) The plants have most likely excreted some of the 14C-glyphosate via their roots. Subsequently, the 14C-glyphosate is (partly) microbially degraded/mineralized—thereby partly incorporated back into microorganisms too—and partly converted to released 14CO2 [55,56,57,58]; various studies have already shown that glyphosate is used by microorganisms in the soil as a carbon and phosphorus source [55,57,58]. We did not investigate this part further. (II) We filtrated the root washing solutions after harvest for LSC measurements. Therefore, we assume to lose adsorbed 14C-glyphosate on solids, namely on soil, but especially on perlite, well known for its huge surface with numerous pores, which was included initially for ventilation purposes in the experimental conception.
No statistically significant differences in measured 14C-glyphosate absorption patterns were found among the Alopecurus myosuroides biotypes tested (Table 2). This is consistent with several studies on other plant species—in many cases, no differences in glyphosate absorption between sensitive and resistant plants have been found [29,59,60,61,62].
The recorded images show clear differences in the 14C-glyphosate translocation pattern between the biotypes studied (Figure 1). In all plants of biotypes S-0 (glyphosate-susceptible) and A1.3 (glyphosate-tolerant), the treated leaves (TLs) contain a very high radioactivity distribution over the entire leaf.
This is consistent with the measured high 14C-glyphosate levels in the treated leaves of all biotypes, ranging from 36.8 to 82.9% of aR. S-0 (Figure 1a,b), which shows only a low degree of translocation of the absorbed 14C-glyphosate into the above-ground plant organs. In both plants, only the pseudostem (PS) of the originally treated leaf is powerful visible in imaging. In Figure 1c, the remaining associated leaves are visible on the pseudostem. In Figure 1a,b, only a marginal 14C-glyphosate translocation took place into certain parts of the roots. In contrast, in Figure 1c, an overall higher translocation into all plant organs is visible. In all plants of A1.3 in Figure 1d–f, a much higher 14C-glyphosate translocation took place into almost all above-ground plant parts. Also, the roots are completely visible with varying degrees of radioactivity in all three plants. The differences visible on the exemplary phosphor images (Figure 1) confirm the differences in measured 14C-glyphosate translocation patterns. In some glyphosate-resistant biotypes, a statistically significant difference in the 14C-glyphosate translocation pattern from the treated leaf to the remaining plant parts, compared to the two S-biotypes, was observed (Table 2). In A1.3, A1.2, and A2.2, significantly more 14C-glyphosate was translocated out of the treated leaf with 52.4, 63.2 and 61.0% of aR, respectively, compared to the sensitive biotype S-0 with 17.1% of aR. In A1.2 and A2.2, also significantly more glyphosate was translocated out of the treated leaf compared to the sensitive biotype S-0125 with 27.5% of aR.
Consequently, significant differences also occurred in the distribution of 14C-glyphosate in the individual plant parts. In biotypes A1.3 and A1.2, 10.9% and 17.6% of aR, respectively, of the originally absorbed 14C-glyphosate was translocated into the untreated leaves (ULs), approximately 3–5 times more than in S-0 with almost 2.8% of aR. Analogously, this was also shown in the comparison between A1.2 and S-0125 (4.6% of aR). The translocation of 14C-glyphosate from TL to PS was more than 2 times higher for A1.2 and A2.2 with 37.3 and 43.7% of aR, respectively, compared to S-0 and S-0125 with only 13.5 and 17.3% of aR, respectively. The translocation from TL to RO was by far the highest in A1.3 compared to all other biotypes. In A1.3, with 25.9% of aR, almost 32 times as much was translocated into the RO than in S-0 with only 0.8% of aR. Furthermore, large differences between the S-0 and the other biotypes are also evident—despite the lack of statistical significance. In all biotypes, in contrast to S-0, a 7- to 30-fold higher amount of 14C-glyphosate was transferred into the RO (Table 2).
The results of the comparison of 14C-glyphosate translocation patterns between the Alopecurus myosuroides biotypes studied here are partly in contrast to the results of other studies [22,25,29,30,31,62,63,64]. In all our glyphosate-resistant biotypes A1.3, A1.2, and A2.2, significantly more 14C-glyphosate was translocated out of the treated leaf into the remaining plant parts than in our two sensitive biotypes S-0 and S-0125. In the mentioned studies, only a reduced translocation of 14C-glyphosate from the treated leaves to the remaining plant parts in resistant plants has always been observed so far. Examples are investigated in, e.g., hairy fleabane [Conyza bonariensis (L.) Cronquist] [63], Canadian horseweed [Conyza canadensis (L.) Cronquist] [64], Italian ryegrass [Lolium multiflorum Lam.] [31], perennial ryegrass [Lolium perenne L.] [29], rescuegrass [Bromus catharticus Vahl] [22], rigid ryegrass [Lolium rigidum Gaudin] [30], and waterhemp [Amaranthus tuberculatus (Moq.) Sauer] [25].

4. Discussion

To investigate glyphosate absorption and translocation patterns in survivors of prior glyphosate application, the methods used in our study differ from those used in other studies: the Alopecurus myosuroides plants studied were (I) obtained by vegetative propagation to obtain sufficient genetically identical plant material compared to obtaining plants from seeds, and, to show more distinct effects, (II) studied at a higher developmental stage, (III) treated with a higher glyphosate dose than the full recommended or permitted glyphosate dose, and (IV) studied with a longer time interval between glyphosate application and the determination of 14C-glyphosate absorption and translocation. Therefore, the comparability with other studies is limited.
The plants also differ macroscopically: A1.3 and A1.2 (938 and 1075 mg, respectively) had produced more than twice as much biomass in contrast to S-0 (401 mg) (Table 2). Consequently, A1.2 and A1.3 probably have also more meristematic tissue in, e.g., the roots (Figure 1) than S-0. Glyphosate is predominantly translocated to metabolic sinks, such as meristematic tissues [9,65], after uptake into the plants. The highest levels of EPSPS are found in the meristems [9]. Consequently, the more meristematic tissues there are in a plant, the higher the translocation of glyphosate into these tissues could be. But, in our experiment, A2.2 formed with 358 mg less biomass than S-0, and S-0125 formed with 1283 mg more biomass compared to A1.2 and A2.2. Therefore, the significant differences cannot be explained by the presence of larger amounts of meristematic tissue.
Regarding the level of 14C-glyphosate translocation out of the treated leaf, it is striking that the translocation was higher in all the biotypes examined compared with S-0. The same applies to the 14C-glyphosate translocation from the treated leaf into the roots. Even S-0125, which together with S-0 originates from the same population, showed a higher 14C-glyphosate translocation. However, the differences between S-0125 and S-0 were not statistically significant. S-0 was the only biotype that was not treated with glyphosate before the experiments described in this study were conducted. This suggests that prior glyphosate exposure may have induced epigenetic changes in the biotypes tested. The different translocation patterns of 14C-glyphosate that we observed may be caused by these assumed epigenetic changes. Epigenetic changes may have altered the regulation of certain genes that influence the transport and location of glyphosate in the plant. A contribution of stress-induced and partly also heritable (stress memory) epigenetic changes in the development of herbicide resistance in weeds is assumed. Here, a herbicide could induce a signal (a regulatory cascade) that triggers changes in gene expression and/or the activity of RNA-mediated DNA methylation. This could lead to an altered regulation of genes that ultimately cause herbicide resistance [43]. In winter wheat [Triticum aestivum L.], different glyphosate concentrations have led to epigenetic changes through an increase in DNA methylation [66]. In mouse ear cress [Arabidopsis thaliana (L.) Heynh.], even different sublethal doses of glyphosate led to epigenetic changes by altering the methylation patterns in this genome [67]. In the study of a glyphosate-resistant and a glyphosate-sensitive C. canadensis population, epigenetic changes due to different methylation patterns between the genomes of the two populations were found after glyphosate treatment [68].
An alternative explanation for the higher degree of 14C-glyphosate translocation in the resistant Alopecurus myosuroides biotypes could be a targeted vacuolar sequestration of glyphosate after translocation into not with 14C-glyphosate-treated tissues. This would limit the amount of glyphosate that reaches the chloroplasts within the cells and thus the site of EPSPS action in the 14C-glyphosate-untreated tissues. Vacuole sequestration of glyphosate has almost always been observed only in leaves treated with glyphosate [8,14,69]. In Conyza canadensis, in addition to vacuole sequestration in glyphosate-treated leaves, vacuole sequestration was also observed in untreated tissues following glyphosate translocation [8,70].
The high accumulation of 14C-glyphosate in the roots observed in A1.3 possibly indicates an increased sequestration of glyphosate into the soil via root exudates, too. Individual root exudations were not investigated in this study. If the increased exudation of glyphosate via the roots plays a role in A1.3 and/or in the other biotypes tested, then 14C-glyphosate absorption via the treated leaf should consequently also have been higher in the biotypes concerned. In this case, the results of the investigation of 14C-glyphosate absorption would not correspond to reality. It is suspected that such a resistance mechanism against herbicides could be caused by overactivity or genetic overexpression of certain transporters like transporter proteins. This includes transporters that cause increased translocation of herbicides via the phloem into the roots beside transporters localized in the roots, which cause increased exudation of herbicides out of the roots. Such a resistance mechanism against glyphosate has not yet been observed in weeds. However, there are weed species such as leafy spurge [Euphorbia esula L.] [71] and Lolium multiflorum [72] that are known to secrete herbicide molecules into the rhizosphere after herbicide treatment [41]. There are also several species of weeds such as quinoa [Chenopodium quinoa Willd.] [73] and tall windmill grass [Chloris elata Desv.] [74] that even secrete glyphosate into the rhizosphere. Two other cases are also known, where the root exudation of herbicides has contributed to herbicide resistance [41]—resistance to imazamox in Mexican fire plant [Euphorbia heterophylla L.] [75] and resistance to MCPA (4-chloro-2-ethylphenoxyacetate) in wild radish [Raphanus raphanistrum L.] [76].
In addition to the hypothetical resistance mechanisms mentioned so far, further mechanisms could also play a role in the resistant biotypes. These include the (I) metabolic degradation of glyphosate by the increased synthesis of a glyphosate-degrading enzyme such as aldo-keto reductase [34]; (II) extrusion of glyphosate from the cellular cytoplasm into the extracellular space by, e.g., an over-regulated ABC-transporter in, e.g., the plasma membrane [37]; (III) mutations in the EPSPS gene, leading to amino acid substitutions in EPSPS at positions other than position 106 [20]; (IV) increased EPSPS gene amplification (gene duplication) [14,21].
It should be noted that no Pro-106 mutations in the EPSPS gene were found among the surviving plants. A possibly present EPSPS gene amplification in the biotypes was not investigated, as no amplification could be found in the Alopecurus myosuroides biotypes in a previous study [51].
The described resistance mechanism of rapid necrosis of glyphosate-treated mature leaf tissue, which results in reduced glyphosate translocation [35,36], can be excluded in the resistant biotypes. The results of our 14C-glyphosate translocation study, combined with the response of the plants in the previous dose–response study [52], are not consistent with the symptoms of such a mechanism.
Suspected resistance mechanisms could result from genetic changes in the resistant Alopecurus myosuroides biotypes studied here or, alternatively, epigenetic changes in the genome could be the reason. The observed different 14C-glyphosate translocation patterns may also be an effect of various physiological processes not yet understood.
The Alopecurus myosuroides populations examined in the dose–response study, from which the biotypes examined here originated, could not yet be classified as glyphosate-resistant [52]. But the surviving and subsequently tested Alopecurus myosuroides biotypes showed resistance to glyphosate. It should be noted that the fulfillment of most of the criteria for herbicide resistance defined by the HRAC [12] has not been investigated for the biotypes tested. These criteria are the following: (I) the fulfillment of the Weed Science Society of America and International Survey of Herbicide-Resistant Weeds definition of resistance; (II) data confirmation of resistance; (III) the resistance must be heritable; (IV) a demonstration of practical field impact [12].

5. Conclusions

Our investigations show the first signs of an incipient development of resistance by finding a few resistant individuals in the tested populations. This assumption is strengthened by the fact that Alopecurus myosuroides has already developed resistance to herbicides with six different mechanisms of action (among them also against glyphosate) [13]. Furthermore, a study [77] provided clear evidence that the prerequisites for the formation of glyphosate resistance in different populations of Alopecurus myosuroides are evident, and that Alopecurus myosuroides populations are undergoing selection for glyphosate resistance in the field. The heritability of reduced sensitivity to glyphosate has been demonstrated. Furthermore, a direct epidemiological link between historical glyphosate selection and current population-level sensitivity has been demonstrated. It has also been shown that current field populations respond to further glyphosate selection.
The results and assumptions presented in this study suggest that further experiments are needed to elucidate the underlying resistance mechanisms in the biotypes of the Alopecurus myosuroides populations from Lower Saxony studied.

Author Contributions

Conceptualization, D.M.W., M.R., D.H. and T.P.; methodology, M.R. and D.H.; validation, M.R. and D.H.; formal analysis, M.R.; investigation, M.R. and D.H.; resources, D.M.W.; data curation, M.R.; writing—original draft preparation, M.R.; writing—review and editing, D.H. and M.R.; visualization, M.R. and D.H.; supervision, D.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest. The funding sponsors (only basic funders) had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

References

  1. Duke, S.O.; Powles, S.B. Glyphosate: A once-in-a-century herbicide. Pest Manag. Sci. 2008, 64, 319–325. [Google Scholar] [CrossRef] [PubMed]
  2. Duke, S.O.; Powles, S.B.; Sammons, R.D. Glyphosate—How it became a once in a hundred-year herbicide and its future. Outlooks Pest Manag. 2018, 29, 247–251. [Google Scholar] [CrossRef]
  3. Benbrook, C.M. Trends in glyphosate herbicide use in the United States and globally. Environ. Sci. Eur. 2016, 28, 3. [Google Scholar] [CrossRef]
  4. Nandula, V.K.; Reddy, K.N.; Koger, C.H.; Poston, D.H.; Rimando, A.M.; Duke, S.O.; Bond, J.A.; Ribeiro, D.N. Multiple Resistance to Glyphosate and Pyrithiobac in Palmer Amaranth (Amaranthus palmeri) from Mississippi and Response to Flumiclorac. Weed Sci. 2012, 60, 179–188. [Google Scholar] [CrossRef]
  5. Powles, S.B.; Yu, Q. Evolution in Action: Plants Resistant to Herbicides. Annu. Rev. Plant Biol. 2010, 61, 317–347. [Google Scholar] [CrossRef]
  6. Green, J.M. Evolution of Glyphosate-Resistant Crop Technology. Weed Sci. 2009, 57, 108–117. [Google Scholar] [CrossRef]
  7. Powles, S.B. Evolved glyphosate-resistant weeds around the world: Lessons to be learnt. Pest Manag. Sci. 2008, 64, 360–365. [Google Scholar] [CrossRef]
  8. D’Avignon, D.A.; Ge, X. In vivo NMR investigations of glyphosate influence on plant metabolism. J. Magn. Reson. 2018, 292, 59–72. [Google Scholar] [CrossRef]
  9. Shaner, D.L. Role of Translocation as a Mechanism of Resistance to Glyphosate. Weed Sci. 2009, 57, 118–123. [Google Scholar] [CrossRef]
  10. Hock, B.; Elstner, E.F. Plant Toxicology, 4th ed.; CRC Press: New York, NY, USA, 2004; pp. 292–296. [Google Scholar]
  11. Powles, S.B.; Lorraine-Colwill, D.F.; Dellow, J.J.; Preston, C. Evolved Resistance to Glyphosate in Rigid Ryegrass (Lolium rigidum) in Australia. Weed Sci. 1998, 46, 604–607. [Google Scholar] [CrossRef]
  12. Heap, I.; Organizer of the “International Survey of Herbicide-Resistant Weeds”. Criteria for Confirmation of Herbicide-Resistant Weeds. Available online: https://hracglobal.com/files/Criteria-for-Confirmation-of-Herbicide-Resistant-Weeds.pdf (accessed on 8 April 2020).
  13. Heap, I. The International Herbicide-Resistant Weed Database. Available online: www.weedscience.org (accessed on 13 April 2025).
  14. Sammons, R.D.; Gaines, T.A. Glyphosate resistance: State of knowledge. Pest Manag. Sci. 2014, 70, 1367–1377. [Google Scholar] [CrossRef] [PubMed]
  15. Shaner, D.L.; Lindenmeyer, R.B.; Ostlie, M.H. What have the mechanisms of resistance to glyphosate taught us? Pest Manag. Sci. 2012, 68, 3–9. [Google Scholar] [CrossRef] [PubMed]
  16. García, M.J.; Palma-Bautista, C.; Rojano-Delgado, A.M.; Bracamonte, E.; Portugal, J.; Alcántara-de la Cruz, R.; De Prado, R. The Triple Amino Acid Substitution TAP-IVS in the EPSPS Gene Confers High Glyphosate Resistance to the Superweed Amaranthus hybridus. Int. J. Mol. Sci. 2019, 20, 2396. [Google Scholar] [CrossRef]
  17. Perotti, V.E.; Larran, A.S.; Palmieri, V.E.; Martinatto, A.K.; Alvarez, C.E.; Tuesca, D.; Permingeat, H.R. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate resistant Amaranthus hybridus population from Argentina. Pest Manag. Sci. 2019, 75, 1242–1251. [Google Scholar] [CrossRef] [PubMed]
  18. Takano, H.K.; Fernandes, V.N.A.; Adegas, F.S.; Oliveira, R.S., Jr.; Westra, P.; Gaines, T.A.; Dayan, F.E. A novel TIPT double mutation in EPSPS conferring glyphosate resistance in tetraploid Bidens subalternans. Pest Manag. Sci. 2020, 76, 95–102. [Google Scholar] [CrossRef] [PubMed]
  19. Yu, Q.; Jalaludin, A.; Han, H.; Chen, M.; Sammons, R.D.; Powles, S.B. Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance. Plant Physiol. 2015, 167, 1440–1447. [Google Scholar] [CrossRef]
  20. Li, J.; Peng, Q.; Han, H.; Nyporko, A.; Kulynych, T.; Yu, Q.; Powles, S. Glyphosate Resistance in Tridax procumbens via a Novel EPSPS Thr-102-Ser Substitution. J. Agric. Food Chem. 2018, 66, 7880–7888. [Google Scholar] [CrossRef]
  21. Gaines, T.A.; Patterson, E.L.; Neve, P. Molecular mechanisms of adaptive evolution revealed by global selection for glyphosate resistance. New Phytol. 2019, 223, 1770–1775. [Google Scholar] [CrossRef]
  22. Yanniccari, M.; Vázquez-García, J.G.; Gómez-Lobato, M.E.; Rojano-Delgado, A.M.; Alves, P.L.d.C.A.; De Prado, R. First Case of Glyphosate Resistance in Bromus catharticus Vahl.: Examination of Endowing Resistance Mechanisms. Front. Plant Sci. 2021, 12, 617945. [Google Scholar] [CrossRef]
  23. Michitte, P.; De Prado, R.; Espinoza, N.; Ruiz-Santaella, J.P.; Gauvrit, C. Mechanisms of Resistance to Glyphosate in a Ryegrass (Lolium multiflorum) Biotype from Chile. Weed Sci. 2007, 55, 435–440. [Google Scholar] [CrossRef]
  24. Nandula, V.K.; Reddy, K.N.; Poston, D.H.; Rimando, A.M.; Duke, S.O. Glyphosate Tolerance Mechanism in Italian Ryegrass (Lolium multiflorum) from Mississippi. Weed Sci. 2008, 56, 344–349. [Google Scholar] [CrossRef]
  25. Nandula, V.K.; Ray, J.D.; Ribeiro, D.N.; Pan, Z.; Reddy, K.N. Glyphosate Resistance in Tall Waterhemp (Amaranthus tuberculatus) from Mississippi is due to both Altered Target-Site and Nontarget-Site Mechanisms. Weed Sci. 2013, 61, 374–383. [Google Scholar] [CrossRef]
  26. Vila-Aiub, M.M.; Balbi, M.C.; Distéfano, A.J.; Fernández, L.; Hopp, E.; Yu, Q.; Powles, S.B. Glyphosate resistance in perennial Sorghum halepense (Johnsongrass), endowed by reduced glyphosate translocation and leaf uptake. Pest Manag. Sci. 2012, 68, 430–436. [Google Scholar] [CrossRef] [PubMed]
  27. Délye, C. Unravelling the genetic bases of non-target-site-based resistance (NTSR) to herbicides: A major challenge for weed science in the forthcoming decade. Pest Manag. Sci. 2013, 69, 176–187. [Google Scholar] [CrossRef]
  28. Alcántara de la Cruz, R.; Barro, F.; Domínguez-Valenzuela, J.A.; De Prado, R. Physiological, morphological and biochemical studies of glyphosate tolerance in Mexican Cologania (Cologania broussonetii (Balb.) DC.). Plant Physiol. Bioch. 2016, 98, 72–80. [Google Scholar] [CrossRef]
  29. Ghanizadeh, H.; Harrington, K.C.; James, T.K.; Woolley, D.J.; Ellison, N.W. Mechanisms of glyphosate resistance in two perennial ryegrass (Lolium perenne) populations. Pest Manag. Sci. 2015, 71, 1617–1622. [Google Scholar] [CrossRef] [PubMed]
  30. Lorraine-Colwill, D.F.; Powles, S.B.; Hawkes, T.R.; Hollinshead, P.H.; Warner, S.A.J.; Preston, C. Investigations into the mechanism of glyphosate resistance in Lolium rigidum. Pestic. Biochem. Phys. 2002, 74, 62–72. [Google Scholar] [CrossRef]
  31. Perez-Jones, A.; Park, K.-W.; Polge, N.; Colquhoun, J.; Mallory-Smith, C.A. Investigating the mechanisms of glyphosate resistance in Lolium multiflorum. Planta 2007, 226, 395–404. [Google Scholar] [CrossRef]
  32. Preston, C.; Wakelin, A.M. Resistance to glyphosate from altered herbicide translocation patterns. Pest Manag. Sci. 2008, 64, 372–376. [Google Scholar] [CrossRef]
  33. Baek, Y.; Bobadilla, L.K.; Giacomini, D.A.; Montgomery, J.S.; Murphy, B.P.; Tranel, P.J. Evolution of Glyphosate-Resistant Weeds. In Reviews of Environmental Contamination and Toxicology; Knaak, J.B., Ed.; Springer: Cham, Switzerland, 2021; Volume 255, pp. 93–128. [Google Scholar]
  34. Pan, L.; Yu, Q.; Han, H.; Mao, L.; Nyporko, A.; Fan, L.; Bai, L.; Powles, S. Aldo-keto Reductase Metabolizes Glyphosate and Confers Glyphosate Resistance in Echinochloa colona. Plant Physiol. 2019, 181, 1519–1534. [Google Scholar] [CrossRef]
  35. Moretti, M.L.; Van Horn, C.R.; Robertson, R.; Segobye, K.; Weller, S.C.; Young, B.G.; Johnson, W.G.; Sammons, R.D.; Wang, D.; Ge, X.; et al. Glyphosate resistance in Ambrosia trifida: Part 2. Rapid response physiology and non-target-site resistance. Pest Manag. Sci. 2018, 74, 1079–1088. [Google Scholar] [CrossRef] [PubMed]
  36. Van Horn, C.R.; Moretti, M.L.; Robertson, R.R.; Segobye, K.; Weller, S.C.; Young, B.G.; Johnson, W.G.; Schulz, B.; Green, A.C.; Jeffery, T.; et al. Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate. Pest Manag. Sci. 2018, 74, 1071–1078. [Google Scholar] [CrossRef]
  37. Pan, L.; Yu, Q.; Wang, J.; Han, H.; Mao, L.; Nyporko, A.; Maguza, A.; Fan, L.; Bai, L.; Powles, S. An ABCC-type transporter endowing glyphosate resistance in plants. Proc. Natl. Acad. Sci. USA 2021, 118, e2100136118. [Google Scholar] [CrossRef] [PubMed]
  38. Amrhein, N.; Martinoia, E. An ABC transporter of the ABCC subfamily localized at the plasma membrane confers glyphosate resistance. Proc. Natl. Acad. Sci. USA 2021, 118, e2104746118. [Google Scholar] [CrossRef] [PubMed]
  39. Cechin, J.; Piasecki, C.; Benemann, D.P.; Kremer, F.S.; Galli, V.; Maia, L.C.; Agostinetto, D.; Vargas, L. Transcriptome Analysis Identifies Candidate Target Genes Involved in Glyphosate-Resistance Mechanism in Lolium multiflorum. Plants 2020, 9, 685. [Google Scholar] [CrossRef]
  40. Gaines, T.A.; Duke, S.O.; Morran, S.; Rigon, C.A.G.; Tranel, P.J.; Küpper, A.; Dayan, F.E. Mechanisms of evolved herbicide resistance. J. Biol. Chem. 2020, 295, 10307–10330. [Google Scholar] [CrossRef]
  41. Ghanizadeh, H.; Harrington, K.C. Perspective: Root exudation of herbicides as a novel mode of herbicide resistance in weeds. Pest Manag. Sci. 2020, 76, 2543–2547. [Google Scholar] [CrossRef]
  42. Kleinman, Z.; Rubin, B. Non-target-site glyphosate resistance in Conyza bonariensis is based on modified subcellular distribution of the herbicide. Pest Manag. Sci. 2017, 73, 246–253. [Google Scholar] [CrossRef]
  43. Markus, C.; Pecinka, A.; Karan, R.; Barney, J.N.; Merotto, A., Jr. Epigenetic regulation—Contribution to herbicide resistance in weeds? Pest Manag. Sci. 2018, 74, 275–281. [Google Scholar] [CrossRef]
  44. Zhang, C.; Yu, C.J.; Yu, Q.; Guo, W.L.; Zhang, T.J.; Tian, X.S. Evolution of multiple target-site resistance mechanisms in individual plants of glyphosate-resistant Eleusine indica from China. Pest Manag. Sci. 2021, 77, 4810–4817. [Google Scholar] [CrossRef]
  45. Augustin, B.; Gehring, K. First glyphosate resistance in Germany. In Proceedings of the 29th German Conference on Weed Biology and Weed Control, Braunschweig, Germany, 3–5 March 2020. [Google Scholar]
  46. Augustin, B.; Gehring, K. Reduced efficacy of glyphosate against annual fescue (Vulpia myuros). In Proceedings of the 29th German Conference on Weed Biology and Weed Control, Braunschweig, Germany, 3–5 March 2020. [Google Scholar]
  47. Augustin, B.; Kunkemöller, M. Glyphosate resistance of Rat’s-tail Fescue (Vulpia myuros). In Proceedings of the 30th German Conference on Weed Biology and Weed Control, Braunschweig, Germany, 22–24 February 2022. [Google Scholar]
  48. Balgheim, N. Investigations on Herbicide Resistant Grass Weeds. Ph.D. Thesis, University of Hohenheim, Stuttgart, Germany, 17 November 2009. [Google Scholar]
  49. Lutman, P.J.W.; Moss, S.R.; Cook, S.; Welham, S.J. A review of the effects of crop agronomy on the management of Alopecurus myosuroides. Weed Res. 2013, 53, 299–313. [Google Scholar] [CrossRef]
  50. Moss, S.R.; Perryman, S.A.M.; Tatnell, L.V. Managing Herbicide-Resistant Blackgrass (Alopecurus myosuroides): Theory and Practice. Weed Technol. 2007, 21, 300–309. [Google Scholar] [CrossRef]
  51. Wolber, D.M.; Warnecke-Busch, G.; Köhler, L.; Kregel, M.; Radziewicz, M. Variability in glyphosate efficacy in Alopecurus myosuroides HUDS. (blackgrass) in Lower Saxony. In Proceedings of the 28th German Conference on Weed Biology and Weed Control, Braunschweig, Germany, 27 February–1 March 2018. [Google Scholar]
  52. Radziewicz, M.; Wolber, D.M.; Warnecke-Busch, G.; Köhler, L.; Hofmann, D.; Pütz, T. Response to Glyphosate in Alopecurus myosuroides Populations from Lower Saxony. In Proceedings of the 29th German Conference on Weed Biology and Weed Control, Braunschweig, Germany, 3–5 March 2020. [Google Scholar]
  53. Meier, U. Growth Stages of Mono- and Dicotyledonous Plants—BBCH Monograph, 2nd ed.; Julius Kühn-Institut (JKI) Federal Research Centre for Cultivated Plants: Quedlinburg, Germany, 2018; pp. 141–146. [Google Scholar]
  54. Koch, M.; Schiedung, H.; Siebers, N.; McGovern, S.; Hofmann, D.; Vereecken, H.; Amelung, W. Quantitative imaging of 33P in plant materials using 14C polymer references. Anal. Bioanal. Chem. 2019, 411, 1253–1260. [Google Scholar] [CrossRef]
  55. Borggaard, O.K.; Gimsing, A.L. Fate of glyphosate in soil and the possibility of leaching to ground and surface waters: A review. Pest Manag. Sci. 2008, 64, 441–456. [Google Scholar] [CrossRef]
  56. Sørensen, S.R.; Schultz, A.; Jacobsen, O.S.; Aamand, J. Sorption, desorption and mineralisation of the herbicides glyphosate and MCPA in samples from two Danish soil and subsurface profiles. Environ. Pollut. 2006, 141, 184–194. [Google Scholar] [CrossRef] [PubMed]
  57. Krzyśko-Łupicka, T.; Orlik, A. The use of glyphosate as the sole source of phosphorus or carbon for the selection of soil-borne fungal strains capable to degrade this herbicide. Chemosphere 1997, 34, 2601–2605. [Google Scholar] [CrossRef]
  58. Shinabarger, D.L.; Braymer, H.D. Glyphosate catabolism by Pseudomonas sp. Strain PG2982. J. Bacteriol. 1986, 168, 702–707. [Google Scholar] [CrossRef]
  59. Alarcón-Reverte, R.; García, A.; Urzúa, J.; Fischer, A.J. Resistance to Glyphosate in Junglerice (Echinochloa colona) from California. Weed Sci. 2013, 61, 48–54. [Google Scholar] [CrossRef]
  60. Bostamam, Y.; Malone, J.M.; Dolman, F.C.; Boutsalis, P.; Preston, C. Rigid Ryegrass (Lolium rigidum) Populations Containing a Target Site Mutation in EPSPS and Reduced Glyphosate Translocation Are More Resistant to Glyphosate. Weed Sci. 2012, 60, 474–479. [Google Scholar] [CrossRef]
  61. Ghanizadeh, H.; Harrington, K.C.; James, T.K.; Woolley, D.J.; Ellison, N.W. Restricted Herbicide Translocation Was Found in Two Glyphosate-resistant Italian Ryegrass (Lolium multiflorum Lam.) Populations from New Zealand. J. Agric. Sci. Tech-Iran 2016, 18, 1041–1051. [Google Scholar]
  62. Nandula, V.K.; Wright, A.A.; Van Horn, C.R.; Molin, W.T.; Westra, P.; Reddy, K.N. Glyphosate Resistance in Giant Ragweed (Ambrosia trifida L.) from Mississippi Is Partly Due to Reduced Translocation. Am. J. Plant Sci. 2015, 6, 2104–2113. [Google Scholar] [CrossRef]
  63. Dinelli, G.; Marotti, I.; Bonetti, A.; Catizone, P.; Urbano, J.M.; Barnes, J. Physiological and molecular bases of glyphosate resistance in Conyza bonariensis biotypes from Spain. Weed Res. 2008, 48, 257–265. [Google Scholar] [CrossRef]
  64. Dinelli, G.; Marotti, I.; Bonetti, A.; Minelli, M.; Catizone, P.; Barnes, J. Physiological and molecular insight on the mechanisms of resistance to glyphosate in Conyza canadensis (L.) Cronq. biotypes. Pestic. Biochem. Phys. 2006, 86, 30–41. [Google Scholar] [CrossRef]
  65. Duke, S.O. The history and current status of glyphosate. Pest Manag. Sci. 2018, 74, 1027–1034. [Google Scholar] [CrossRef]
  66. Nardemir, G.; Agar, G.; Arslan, E.; Erturk, F.A. Determination of genetic and epigenetic effects of glyphosate on Triticum aestivum with RAPD and CRED-RA techniques. Theor. Exp. Plant Phys. 2015, 27, 131–139. [Google Scholar] [CrossRef]
  67. Kim, G.; Clarke, C.R.; Larose, H.; Tran, H.T.; Haak, D.C.; Zhang, L.; Askew, S.; Barney, J.; Westwood, J.H. Herbicide injury induces DNA methylome alterations in Arabidopsis. PeerJ 2017, 5, e3560. [Google Scholar] [CrossRef] [PubMed]
  68. Margaritopoulou, T.; Tani, E.; Chachalis, D.; Travlos, I. Involvement of Epigenetic Mechanisms in Herbicide Resistance: The Case of Conyza canadensis. Agriculture 2018, 8, 17. [Google Scholar] [CrossRef]
  69. Ge, X.; d’Avignon, D.A.; Ackerman, J.J.H.; Collavo, A.; Sattin, M.; Ostrander, E.L.; Hall, E.L.; Sammons, R.D.; Preston, C. Vacuolar Glyphosate-Sequestration Correlates with Glyphosate Resistance in Ryegrass (Lolium spp.) from Australia, South America, and Europe: A 31P NMR Investigation. J. Agric. Food Chem. 2012, 60, 1243–1250. [Google Scholar] [CrossRef]
  70. Ge, X.; D’Avignon, D.A.; Ackerman, J.J.H.; Sammons, R.D. Rapid vacuolar sequestration: The horseweed glyphosate resistance mechanism. Pest Manag. Sci. 2010, 66, 345–348. [Google Scholar] [CrossRef]
  71. Hickman, M.V.; Messersmith, C.G.; Lym, R.G. Picloram release from leafy spurge roots. J. Range Manag. 1990, 43, 442–445. [Google Scholar] [CrossRef]
  72. Dinelli, G.; Bonetti, A.; Marotti, I.; Minelli, M.; Busi, S.; Catizone, P. Root exudation of diclofop-methyl and triasulfuron from foliar-treated durum wheat and ryegrass. Weed Res. 2007, 47, 25–33. [Google Scholar] [CrossRef]
  73. Laitinen, P.; Rämö, S.; Siimes, K. Glyphosate translocation from plants to soil—Does this constitute a significant proportion of residues in soil? Plant Soil 2007, 300, 51–60. [Google Scholar] [CrossRef]
  74. Brunharo, C.A.C.G.; Patterson, E.L.; Carrijo, D.R.; de Melo, M.S.C.; Nicolai, M.; Gaines, T.A.; Nissen, S.J.; Christoffoleti, P.J. Confirmation and mechanism of glyphosate resistance in tall windmill grass (Chloris elata) from Brazil. Pest Manag. Sci. 2016, 72, 1758–1764. [Google Scholar] [CrossRef]
  75. Rojano-Delgado, A.M.; Portugal, J.M.; Palma-Bautista, C.; Alcántara-de la Cruz, R.; Torra, J.; Alcántara, E.; De Prado, R. Target site as the main mechanism of resistance to imazamox in a Euphorbia. heterophylla. biotype. Sci. Rep. 2019, 9, 15423. [Google Scholar] [CrossRef] [PubMed]
  76. Jugulam, M.; DiMeo, N.; Veldhuis, L.J.; Walsh, M.; Hall, J.C. Investigation of MCPA (4-Chloro-2-ethylphenoxyacetate) Resistance in Wild Radish (Raphanus raphanistrum L.). J. Agric. Food Chem. 2013, 61, 12516–12521. [Google Scholar] [CrossRef]
  77. Comont, D.; Hicks, H.; Crook, L.; Hull, R.; Cocciantelli, E.; Hadfield, J.; Childs, D.; Freckleton, R.; Neve, P. Evolutionary epidemiology predicts the emergence of glyphosate resistance in a major agricultural weed. New Phytol. 2019, 223, 1584–1594. [Google Scholar] [CrossRef]
Ijpb 16 00045 g001
Figure 1. Plants with corresponding phosphor images of glyphosate-susceptible S-0 ((a–c), 264 h HAT) and -tolerant A1.3 ((d–f), 264 h HAT) Alopecurus myosuroides biotypes, respectively. Image plates are expressed in full size (350 mm × 500 mm); photographs are zoomed for better detail visualization. Example images between S and A1.3 showed large differences in the translocation pattern. Treated leaf is always illustrated in the top right. Red color indicates low activity and yellow, green, blue color indicates raising activities, respectively, white is overexpressed.
Figure 1. Plants with corresponding phosphor images of glyphosate-susceptible S-0 ((a–c), 264 h HAT) and -tolerant A1.3 ((d–f), 264 h HAT) Alopecurus myosuroides biotypes, respectively. Image plates are expressed in full size (350 mm × 500 mm); photographs are zoomed for better detail visualization. Example images between S and A1.3 showed large differences in the translocation pattern. Treated leaf is always illustrated in the top right. Red color indicates low activity and yellow, green, blue color indicates raising activities, respectively, white is overexpressed.
Ijpb 16 00045 g001
Table 1. Alopecurus myosuroides plants used for the examination of 14C-glyphosate absorption and translocation with previously survived glyphosate dose and BBCH-stage at the time of propagation.
Table 1. Alopecurus myosuroides plants used for the examination of 14C-glyphosate absorption and translocation with previously survived glyphosate dose and BBCH-stage at the time of propagation.
Plant Clones from PopulationSurvival at Previous Dose Rate
(g a.i. ha−1)		Growth Stage aBBCH Stage No.Replications
S-00Beginning of shooting—1 node stage30–313
S-01252259 or more tillers293
P1118009 or more tillers292
P3918009 or more tillers293
A1.318007 tillers273
A1.236009 or more tillers292
A2.23600Beginning of shooting—1 node stage30–313
a According to the Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie (BBCH)-scale for weeds [53].
Table 2. Mean plant weight after oven drying, absorption, translocation (radioactivity outside of TL), and distribution (radioactivity in TL, UL, PS, and RO) of 14C-glyphosate after application on glyphosate-susceptible and -tolerant Alopecurus myosuroides biotypes a, b, c, d.
Table 2. Mean plant weight after oven drying, absorption, translocation (radioactivity outside of TL), and distribution (radioactivity in TL, UL, PS, and RO) of 14C-glyphosate after application on glyphosate-susceptible and -tolerant Alopecurus myosuroides biotypes a, b, c, d.
14C-glyphosate Distribution
Plants (Clones) from IndividualsHATBiomass Mean Plant Dry Weight14C-glyphosate Absorption14C-glyphosate TranslocationTLULPSRO
hmg% Applied% Absorbed
sensitive
S-0		264401 ± 11279 ± 517 ± 13a83 ± 13a3 ± 2ad14 ± 11a0.8 ± 0.4a
sensitive
S-0125		2401283 ± 37181 ± 928 ± 22ab73 ± 22ab5 ± 4abd17 ± 14a5.6 ± 6.1ab
tolerant P11240699 ± 5476 ± 127 ± 8ab73 ± 8ab4 ± 4abd16 ± 5a7.3 ± 8.6ab
tolerant P392401739 ± 13180 ± 1523 ± 7ab78 ± 7ab3 ± 0abd10 ± 7a8.7 ± 6.8ab
tolerant A1.3264938 ± 15877 ± 252 ± 32bcd48 ± 32bc11 ± 6bcd16 ± 10a26 ± 28b
tolerant A1.22641075 ± 6076 ± 1063 ± 15cd37 ± 15c18 ± 9c37 ± 1b8.3 ± 5.3ab
tolerant A2.2240358 ± 4077 ± 961 ± 8cd39 ± 8c8 ± 4d44 ± 9b9.2 ± 2.7ab
p-value 0.98740.0418 0.0418 0.0344 0.0085 0.049
a Abbreviations: HAT, hours after treatment; TL, treated leaf; UL, untreated leaves; PS, pseudostem; RO, roots. b Similar and different letters indicate no difference and significant difference, respectively, between mean values within each column according to Fisher’s LSD tests at 5% level of probability. c Mean values ± standard deviation of the mean. d Radioactivity from root exudates has not been determined for each individual plant—on average for each plant, 8.5% can be assumed.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Radziewicz, M.; Wolber, D.M.; Pütz, T.; Hofmann, D. Altered Translocation Pattern as Potential Glyphosate Resistance Mechanism in Blackgrass (Alopecurus myosuroides) Populations from Lower Saxony. Int. J. Plant Biol. 2025, 16, 45. https://doi.org/10.3390/ijpb16020045

AMA Style

Radziewicz M, Wolber DM, Pütz T, Hofmann D. Altered Translocation Pattern as Potential Glyphosate Resistance Mechanism in Blackgrass (Alopecurus myosuroides) Populations from Lower Saxony. International Journal of Plant Biology. 2025; 16(2):45. https://doi.org/10.3390/ijpb16020045

Chicago/Turabian Style

Radziewicz, Markus, Dirk M. Wolber, Thomas Pütz, and Diana Hofmann. 2025. "Altered Translocation Pattern as Potential Glyphosate Resistance Mechanism in Blackgrass (Alopecurus myosuroides) Populations from Lower Saxony" International Journal of Plant Biology 16, no. 2: 45. https://doi.org/10.3390/ijpb16020045

APA Style

Radziewicz, M., Wolber, D. M., Pütz, T., & Hofmann, D. (2025). Altered Translocation Pattern as Potential Glyphosate Resistance Mechanism in Blackgrass (Alopecurus myosuroides) Populations from Lower Saxony. International Journal of Plant Biology, 16(2), 45. https://doi.org/10.3390/ijpb16020045

Article Metrics

Back to TopTop