Browsing by Subject "Glyphosat"
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Publication Adaptation of model organisms and environmental bacilli to glyphosate gives insight to species-specific peculiarities of the shikimate pathway(2024) Schwedt, Inge; Commichau, FabianGlyphosate (GS), the active ingredient of the popular herbicide Roundup, inhibits the 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase of the shikimate pathway, which is present in archaea, bacteria, Apicomplexa, algae, fungi, and plants. In these organisms, the shikimate pathway is essential for de novo synthesis of aromatic amino acids, folates, quinones and other metabolites. Therefore, the GS-dependent inhibition of the EPSP synthase results in cell death. Previously, it has been observed that isolates of the soil bacteria Burkholderia anthina and Burkholderia cenocepacia are resistant to high amounts of GS. In the framework of this PhD thesis, it could be demonstrated that B. anthina isolates are not intrinsically resistant to GS. However, B. anthina rapidly adapts to the herbicide at the genome level and the characterization of GS-resistant suppressor mutants led to the discovery of a novel GS resistance mechanism. In B. anthina, the acquisition of loss-of-function mutations in the ppsR gene increases GS resistance. The ppsR gene encodes a regulator of the phosphoenolpyruvate (PEP) synthetase PpsA. In the absence of a functional PpsR protein, the bacteria synthesize more PEP, which competes with GS for binding in the active site of the EPSP synthase, increasing GS resistance. The EPSP synthase in B. anthina probably does not allow changes in the amino acid sequence as it is the case in other organisms. Indeed, the Gram-negative model organism Escherichia coli evolves GS resistance by the acquisition of mutations that either reduce the sensitivity of the EPSP synthase or increase the cellular concentration of the enzyme. Unlike E. coli, the EPSP synthase is also critical for the viability of Gram-positive model bacterium Bacillus subtilis. This observation is surprising because the enzyme belongs to the class of GS-insensitive EPSP synthases. In fact, the EPSP synthase is essential for growth of B. subtilis. The determination of the nutritional requirements allowing the growth of B. subtilis and E. coli mutants lacking EPSP synthase activity revealed that the demand for shikimate pathway intermediates is higher in the former organism. This finding explains why laboratory as well as environmental Bacilli exclusively adapt to GS by the mutational inactivation of glutamate transporter genes. Here, it was also shown that a B. subtilis mutant lacking EPSP synthase activity grows in minimal medium only when additional mutations accumulate in genes involved in the regulation of aerobic/anaerobic metabolism and central carbon metabolism. The characterization of these additional mutants will help to elucidate the peculiarities of the shikimate pathway in B. subtilis. Moreover, the mutants could be useful to identify the aromatic amino acid transporters that still await their discovery.Publication Glyphosate use in agro-ecosystems : identification of key factors for a better risk assessment(2010) Tesfamariam, Tsehaye; Römheld, VolkerGlyphosate ([N-phosphonomethyl] glycine) is a non-selective, post-emergence, organo-phosphorous, broad-spectrum herbicide used worldwide for controlling weeds in horticulture, agriculture, silviculture, and urban landscapes. It effectively controls most annual and perennial weed species and is the world´s biggest-selling herbicide. One reason for the popularity of glyphosate is its effect on roots and rhizome systems of weed following foliar application. After coming in contact with soil, glyphosate will be strongly adsorbed and this sorption behavior makes glyphosate unique as compared to most other herbicides and has elicited a general belief that it is rapidly adsorbed to the soil without any residual effect. However, glyphosate adsorption to the soil matrix seems a reversible process and glyphosate conserved in roots of treated target plants has been overlooked in most previous risk assessments. Therefore, in face of the increasing number of yet unexplained observations of negative side effects after glyphosate application, this thesis was initiated to identify possible risk factors associated with the frequent use of glyphosate in agro-ecosystems. For this purpose: (1) relevance of waiting time between weed desiccation by glyphosate and subsequent crop planting, (2) remobilization risk of glyphosate fixed in the soil matrix mediated by pH change in the rhizosphere, (3) glyphosate preservation in target plant roots and (4) contribution of glyphosate released from decaying weed residues for intoxication of following non-target plants were investigated in controlled greenhouse conditions using two contrasting soils: a weakly buffered acidic Arenosol (top soil) and a highly buffered calcareous Luvisol (subsoil). Furthermore, field experiment was conducted to partially confirm the found results of controlled model experiments under greenhouse conditions. These model experiments as well as the experiment in farmer´s field revealed that the residual toxicity of glyphosate increased with a declining waiting time between glyphosate weed desiccation and subsequent crop planting. In the greenhouse experiments, seedling growth and biomass production of sunflower plants were strongly impaired by pre-sowing application of glyphosate in the variants with less than 21 days waiting time. The inhibitory effects on seedling growth were associated with a corresponding increase of shikimate accumulation in the root tissue as physiological indicator for glyphosate toxicity and impairment of the manganes-nutritional status of the sunflower seedlings. Results of the field experiment at Hirrlingen/Tübingen confirmed the relevance of waiting time. Stunted development and heterogeneous emergence of winter wheat plants occurred at field plots where the wheat sowing was done 2 days, compared to plants sown 14 days after foliar application of glyphosate to weed plants. At a short waiting time (2 d), data on visual scoring showed up to 50% of the culture damage that was visually persistent still after 6 months at harvests. This was also associated with a reduced nutritional status of wheat plants Ca, Mg, Zn and Cu, particularly expressed when glyphosate application rate was elevated from 2L to 6 L ha-1. Since glyphosate shows a similar pattern of reaction like that of phosphate in soil, it has been hypothesized that rhizosphere processes responsible for P mobilization are likely to co-mobilize also glyphosate. To test this hypothesis, an experiment was conducted using the two soils with contrasting properties pre-incubated with different rates of glyphosate and supplied with stabilized NH4+-N or NO3--N to induce the different changes in rhizosphere pH. From the results of this experiment, however, it was not possible to confirm this hypothesis. No glyphosate phytotoxicity of sunflower seedlings on the Luvisol with NH4+ could be detected due to observed minor rhizosphere acidification. In agreement, also no shikimate accumulation in root was measured. However, there was a distinct decrease in biomass of the sunflower seedlings at NH4- supply, possibly due to a missing NO3- signal. In contrast in the Arenosol no difference in growth could be shown between both supplied N-forms despite a clearly expressed difference in rhizosphere pH. Root exudation of organic carboxylates has also been considered to assists the release of adsorbed phosphate in the rhizosphere from the soil matrix via exchange chelation. A similar phenomenon was expected for glyphosate. In the present study, however, supplementation of Na-citrate or citric acid to both contrasting soils, pre-incubated with different levels of glyphosate, did not show a clear evidence for an adequate glyphosate remobilization and the subsequent plant damage. On the acidic Arenosol, there was no difference in growth of sunflower seedlings between the treatments. In contrast, on the Luvisol, supplementation of Na citrate (10µmol g-1 soil) but not citric acid indicated some promotion of root growth on glyphosate free treatment. This could not be easily explained because no intracellular shikimate accumulation as bio-indicator for glyphosate could be detected in the treatments with glyphosate pre-incubated soil. In many plant species, glyphosate is not readily metabolized, but preferentially translocated to young growing tissues of roots and shoots, where it can be accumulated in millimolar concentrations. In soil-grown target plants, this inhomogeneous distribution of glyphosate within the root tissues may lead to the formation of hot spots of glyphosate containing root residues in soils. Subsequently this stored glyphosate as hot spots can be released during microbial degradation of root material. To evaluate the potential of roots of target plant in stabilization and subsequent release of glyphosate with intoxication of subsequent crop plants, model experiments were conducted with application of glyphosate either via rye grass as target weed plants or directly to the soil. Sunflower seeds were sown at different waiting times (0-21 days) for both glyphosate application modes. Toxicity of glyphosate applied shortly before sowing of sunflower as non-target was strongly dependent on the mode of glyphosate application. When glyphosate was sprayed on pre-cultured rye grass seedlings as model weed, detrimental effects on plant growth and the Mn nutritional status, as well as increased intracellular shikimate accumulation in root tissue were more strongly expressed than at a direct soil application of the same amount of glyphosate. The increased extent of toxicity after a glyphosate pre-sowing application to pre-cultured rye grass compared with a direct soil application might indicate that the root tissue of glyphosate-treated weeds represents a storage pool for glyphosate in the pots. The globally increasing adoption of no-till or reduced tillage systems are becoming a driving force for an increase of glyphosate use. In such systems, glyphosate is applied pre-sowing for weed control and glyphosate may remain in root and shoot residues. Usually in these reduced tillage systems, only a minimal soil disturbance occurs at sowing, which might lead to limited incorporation of the glyphosate contaminated straw to the upper soil layer where germination of following non-target crop will take place. To evaluate such risk, a pot experiment was conducted under controlled greenhouse conditions with the two contrasting soils. Glyphosate was supplied via glyphosate pre-treated shoot or root material of rye grass applied either as chopped plant material ?straw? or as homogenate. Analysis of physiological parameters such as intracellular shikimate accumulation as metabolic indicator for glyphosate toxicity, biomass production and micronutrient status revealed, that a detrimental effect could be only with treated rye grass shoot material as straw or homogenates incorporated into the Arenosol but not into the Luvisol. This is most probably related to the difference in soil property between the two soils. At this level of glyphosate supply, the detoxification capacity of the highly buffered calcareous subsoil might have played a primary role in preventing glyphosate toxicity, while this glyphosate supply level seems beyond the detoxification capacity of the weakly buffered acidic Arenosol. All together, the achieved results of the model pot experiments are in correspondence with that of the reported field experiments. Further, the results revealed the important role of glyphosate stored in root and shoots of weed plants as a glyphosate pool in soils for intoxication of following crops. More information on transformation of these glyphosate enriched crop residues and its glyphosate release during microbial decomposition in different soils are urgently needed for a better precaution and risk assessment of glyphosate use for weed control for farmer´s practice.Publication Re-plant problems in long-term no-tillage cropping systems : causal analysis and mitigation strategies(2016) Afzal; Neumann, GünterNo-tillage is considered as a promising alternative for tillage-based conventional farming, by saving energy-input and time, reducing groundwater pollution and counteracting soil erosion and losses of the soil-organic matter. However, in the recent past, no-tillage farmers in Southwest Germany repeatedly reported problems particularly in winter wheat production, characterized by stunted plant growth in early spring, chlorosis, impaired fine root development and increased disease susceptibility. These symptoms were particularly apparent on field sites with long-term (≥ 10 years) no-tillage history (LT) but not on adjacent short-term (≤ 2 years) no-tillage plots (ST). The effects could be reproduced in pot experiments under controlled conditions, with soils collected from the respective field sites in five different locations, providing a basis for causal analysis. The expression of damage symptoms in pot experiments with sieved soils, excluded differences in soil compaction, induced by long-term no-tillage farming as a potential cause. Soil analysis revealed higher levels of soil organic matter in the topsoil, as expected for LT field sites and no apparent mineral nutrient deficiencies, both, on LT and ST soils. However, phosphate (P) deficiency was characteristic for plants grown on LT soils. Obviously, this was caused by the limited acquisition of sparingly soluble soil P, due to impaired root development but not by low P availability on LT soils. In four out of five cases, gamma-ray soil sterilization did not affect the expression of plant damage symptoms on LT soils, excluding pathogen effects as a major cause. Soil application of biochar, at a rate of 5% (v/v), rapidly restored plant growth on LT soils, detectable already during the first week after sowing. This finding points to the presence of a phytotoxic compound since binding of soil xenobiotics by biochar is well documented. Accumulation of allelopathic compounds, originating from crop residues and root exudates remaining in the topsoil, is a problem related to no-tillage farming, particularly in cases of limited crop rotations or in monocultures, which also applied to the investigated field sites. However, a specific wheat auto-allelopathic effect is unlikely, since similar crop damage was also observed in soybean, sunflower, oilseed rape and various cover crops. Typical for allelopathic effects, in the pot experiments, plant damage symptoms in winter wheat appeared rapidly during emergence and early seedling development. However, under field conditions, germination and early growth were usually not affected, and symptoms were first detectable during re-growth in early spring. Moreover, damage symptoms disappeared when soil sampling was performed in summer instead of early spring, suggesting degradation of the toxic compound, which is also not compatible with the hypothesis of long-term accumulation of allelopathic compounds. The observed temporal pattern of plant damage rather resembled residual effects, occasionally observed after application of certain herbicides with soil activity (e.g., sulfonylureas, propyzamide). Therefore, a systematic survey of herbicide residues was conducted for topsoils on six pairs of LT and ST-field sites. Characteristic for no-tillage farming, glyphosate was the only herbicide, commonly and regularly used on all investigated field sites. The soil analysis revealed higher levels of glyphosate residues on all investigated LT, soils as compared with directly neighboured ST plots. Particularly on LT plots with strong expression of plant damage symptoms, high concentrations of glyphosate (2-4 mg kg-1 soil), and of its metabolite AMPA were detected in the 10 cm topsoil layer. This concentration range is characteristic for residual levels, usually observed several days after glyphosate applications but was still detectable in early spring, six months after the last glyphosate treatment, while only trace concentrations below the detection limit (0.05 mg kg-1 soil) were found in ST soils. Coinciding with the declining plant damage potential, residual glyphosate and AMPA concentrations on LT plots declined during the vegetation period until early summer. No comparable pattern was detectable for residues of other herbicides, such as pendimethalin and propyzamide. Degradation of glyphosate residues in soils correlates with microbial activity. Accordingly, reduced soil respiration as an indicator for microbial activity was detected in four out of five cases in soil samples collected from LT field sites, suggesting delayed glyphosate degradation as compared with ST plots. Due to rapid adsorption, glyphosate usually exhibits extremely limited soil activity. However, at least trace concentrations of glyphosate and AMPA (1.5-3.5 µg L-1) were detectable also in the potentially plant-available, water-soluble phase in spring samples, collected from LT field plots with high potential for plant damage. Nutrient solution experiments, with 3-6 weeks exposure of winter wheat to the residual herbicide concentrations detected in the LT soil solution, revealed the development of chlorosis and similar to soil experiments, a 30%-50% reduction in fine root production, which surprisingly was mainly induced by AMPA and to a lesser extent by glyphosate itself. Accordingly, both, in hydroponics and LT soil experiments, the plant damage symptoms were not associated with shikimate accumulation in the root tissue as a physiological indicator for glyphosate but not for AMPA toxicity. The dominant role of AMPA toxicity also became apparent by the fact that, both, glyphosate resistant (GR) and non-resistant (NR) soybean plants were affected on LT no-tillage soils since transgenic GR plants are not resistant to AMPA. A preliminary RNAseq gene expression analysis of the root tissue just prior to the appearance of visible plant damage symptoms, revealed down-regulation of genes involved in general stress responses, down-regulation of aquaporin genes (PIPs and TIPs) with functions in water uptake and root elongation, down-regulation of ethylene-related genes but up-regulation of cytokinin-related gene expression indicating interferences with hormonal balances. These changes in gene expression patterns relative to the untreated control were detected in plants treated with AMPA and glyphosate+AMPA but not with glyphosate alone. The findings suggest that long-term exposure to subtoxic levels of AMPA, as major glyphosate metabolite temporally accumulated in LT no-tillage soils, can finally interfere with metabolic processes essential for normal root development. A series of pot and field experiments were initiated to test the potential of selected commercial formulations of plant growth-promoting microorganisms, based on strains of Pseudomonas sp., Bacillus amyloliquefaciens, and Trichoderma harzianum, for mitigation of plant stress symptoms, expressed on LT no-tillage field sites in spring. For members of the selected microbial genera, root growth-promoting effects, pathogen suppression, and glyphosate degradation potential have been reported. Unfortunately, plant growth promotion was detectable only on ST soils but was not successful on LT plots, both, in pot and field experiments, probably related to limited root development for microbial colonization and early summer drought under field conditions. As an alternative approach, incorporation of pyrolysis biochar from woody substrates at a rate of 5 % (v/v) to the top 10 cm soil layer of LT soils, equivalent to approx. 35 t ha-1, were able to restore plant growth completely in pot experiments and protected wheat plants from glyphosate overdose applications (up to 8 L Roundup Ultramax® ha-1), even on artificial substrates with low potential for glyphosate adsorption. As a short-term mitigation strategy, field-testing with different biochar concentrations is recommended. During the last two years, farmers also modified their no-tillage management strategies on the investigated field sites by introducing more variable crop rotations including, winter wheat, winter rape, maize and soybean and using mustard, pea, and Crotalaria as cover crops. Despite further annual applications of glyphosate (3 L ha-1 of a 360 g ai L-1 formulation), plant performance on the respective field sites was significantly improved. These observations suggest that limited crop rotation favored the development of a soil microflora with low degradation potential for glyphosate, leading to a decline in degradation rates of glyphosate soil residues and underline the importance of crop diversity management.Publication Rhizosphere processes as determinants for glyphosate damage of non-target plants(2010) Bott, Sebastian; Neumann, GünterDue to low production costs and high herbicidal efficiency, glyphosate is the most widely used wide-spectrum herbicide. Glyphosate acts as a non-selective, total herbicide by inhibiting the biosynthesis of aromatic amino acids. Apart from glyphosate drift contamination, risks of glyphosate toxicity to crop plants and other non-target organisms are generally considered as marginal, because glyphosate is almost instantaneously inactivated by adsorption to the soil matrix and rapid microbial/chemical degradation in the soil solution. However, in the recent past, an increasing number of yet unexplained observations on significant damage of crop plants have been reported in the literature and by farmers, suggesting gaps in the risk assessment, with respect to the fate glyphosate in the rhizosphere and the interaction with rhizosphere processes. According to these observations, the aim of present study was a systematic evaluation of potential rhizosphere effects of glyphosate, including direct toxicity, risks of re-mobilisation by fertiliser application, potential role of pathogens and allelopathic compounds, and interactions with micronutrients, both in glyphosate-sensitive and transgenic glyphosate-resistant crops. A series of field trials in reduced soil tillage cropping systems as well as green-house experiments on soils with contrasting properties with sunflower, winter wheat and soybean, consistently revealed a close clausal relationship between crop damage and (a) short waiting times between glyphosate application on target weeds and subsequent sowing of crops and (b) the density and speed of decay of glyphosate-treated weeds. The results suggested that damage of crop plants is induced by a rhizosphere transfer of glyphosate from weeds to subsequently sown crops. This transfer might take place by contact contamination due to exudation of glyphosate from living roots of treated weeds and/or release during decomposition of the root residues. A comparison between phytotoxic effects of glyphosate and aminomethylphosphonic acid (AMPA) as major metabolite of glyphosate in soils, revealed high toxicity in case of root exposure to glyphosate, but not to AMPA. By contrast, a significant decline of germination was induced by seed exposure to AMPA, while germination was not affected by glyphosate treatments. The observed differences in sensitivity to glyphosate and AMPA in different stages of plant development may explain variable symptoms of crop damage under field conditions, ranging from growth depressions and chlorosis to reduced field emergence. The results of the present study further suggest that risks for crop damage associated with rhizosphere transfer of glyphosate are additionally influenced by a range of environmental factors, such as growth season (spring or fall application), temperature, soil moisture, redox potential of soils and soil microbial activity. These factors might shorten or prolongate the time window for crop damage of glyphosate contact contamination in the rhizosphere under field conditions. Model experiments investigating the sensitivity of different plant species to glyphosate root exposure, revealed significant differences between winter wheat, maize and soybean in terms of glyphosate-induced plant damage but also in their ability for recovery from glyphosate damage suggesting marked genotypic differences in the expression of damage symptoms also under field conditions. In agreement with previous investigations, results of the present study indicated a rapid inactivation of glyphosate by adsorption to the soil matrix. Glyphosate adsorption in soils seem to be mainly mediated by the phosphonate group of the molecule in a way similar to the adsorption of inorganic phosphate. Accordingly glyphosate re-mobilisation is possible via ligand exchange by phosphate application. The results of the present study have demonstrated for the first time that depending on soil properties also the application of fertiliser phosphate is able to re-mobilise glyphosate in sufficient quantities to mediate crop damage in pot experiments. This finding suggest, that re-mobilisation of glyphosate potentially by fertiliser P or root-induced chemical modifications for P and Fe mobilisation needs to be considered as additional potential rhizosphere pathway for glyphosate damage to non-target plants. Field trials and model experiments under soil and hydroponic conditions consistently revealed a significantly impaired nutritional status of glyphosate-sensitive but also glyphosate-resistant crops. However, depending on the culture conditions different mineral nutrients were affected by the glyphosate treatments and plant damage was not related with a certain nutrient deficiency. These findings suggest that damaged root growth, induced by glyphosate toxicity, rather than specific interactions with certain mineral nutrients are responsible for the observed impairment of nutrient acquisition. In conclusion, results of the present study highlight that risks for crop damage associated with glyphosate toxicity in the rhizosphere can be substantial and is influenced by factors such as waiting time after herbicide application, weed density, cropping systems, fertilizer management, genotypic differences, and probably also environmental factors including temperature, soil moisture, and soil microbial activity. The independency between these factors is so far not entirely clear but should be investigated in future studies. Nevertheless, results of present study suggest that risks could be minimized by simple management tools such as the consideration of waiting times between application of glyphosate and sowing of crops particularly in case of high weed densities and alternation of herbicides to reduce not only risk for remobilization of glyphosate but also problems associated to the selection of glyphosate-resistant weeds.