Browsing by Subject "Herbicide resistance"

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Distribution, detection and genetic background of herbicide-resistant Alopecurus myosuroides (Huds.) in Germany
(2015) Rosenhauer, Maria; Gerhards, Roland
Weed control is an important part in agricultural practice. Since selective herbicides were introduced, the labour-intensive mechanical weed control was replaced by chemicals. The use of chemicals for weed control has become increasingly problematic due to the evolution of herbicide-resistant weeds. In Germany, Alopecurus myosuroides (Huds.) is one of the most problematic weeds concerning herbicide resistance. The first resistant black-grass biotype in Germany was found in 1982. More than 30 years after the first resistant black-grass was found in Germany (1982), there are still numerous unsolved questions and challenges concerning the problem of herbicide resistance. Further knowledge about the distribution, the detection, and the genetic background of different resistance mechanisms is needed to find comprehensive solutions for the future. Knowledge about the occurrence and distribution of herbicide-resistant black-grass in Germany, and the herbicides primarily affected may provide more detailed information for farmers to quickly react on upcoming resistances. Moreover, if the genetic background of resistance is better understood, practical conclusions regarding the choice of herbicides and management tools can be drawn. Furthermore, a reliable and easy-to-handle test system for the detection of resistances would enable farmers to react faster and in a more targeted manner. The aim of the present study was to investigate these aspects addressing herbicide-resistant black-grass in Germany. How widespread is TSR in Germany? Did the amount of TSR change over the years? Are there “TSR-hot spots” in Germany? The first paper addressed ACCase and ALS-resistant black-grass samples in Germany. It dealt with the distribution and development of TSR in Germany over a period of nine years. It could be demonstrated that TSR was more widespread than expected. The frequency of ACCase-TSR increased from 4.0% in 2004 to 38.5% in 2012. ALS-TSR rose from 0.8% in 2007 to 13.9% in 2012. Both TSRs significantly increased over time within a standing number of seed samples over the years. How many genes are involved in NTSR? Are there differences between the plants and can we detect cross-resistances? The second paper dealt with the inheritance of NTSR conferring resistance to chlorotoluron, fenoxaprop-P-ethyl, pinoxaden, mesosulfuron + iodosulfuron, and flufenacet in six different black-grass plants. Segregation analyses of the quantitative trait showed a minimum of five loci conferring specific resistances. The resistances against chlorotoluron and fenoxaprop-P-ethyl were mostly conferred by one locus, whereas resistances against pinoxaden and mesosulfuron + iodosulfuron were mostly conferred by a minimum of two loci. A minimum of one to three loci explained resistance to flufenacet. The accumulation of resistance loci in individual plants could be achieved by the study. Furthermore, the number of loci was shown to be herbicide- and plant-specific which further confirms the complexity of NTSR. How is it possible to test pre-emergence herbicides in black-grass? Which test system is the most reliable? The aim of the third paper was to find a reliable test system to monitor pre-emergence herbicide resistance in black-grass. It is widely known that diverse sites of action can be affected by NTSR. Moreover, resistance against pre-emergence herbicides belonging to the HRAC groups N, K3, and C2 occur. The outcomes of the study indicate that a soil-based greenhouse test with pre-germinated seeds is most suitable for resistance detection. Discriminating herbicide rates which were able to distinguish between the resistant and susceptible black-grass biotypes were found for all of the herbicides tested. This enables a reliable, quick, and easy way to identify pre-emergence resistance. In conclusion, herbicide-resistant black-grass has become an increasing problem in Germany. The high frequency of nearly 40% ACCase TSR on resistance suspected sites highlights the importance of changes in agricultural practices. The aim should be to avoid the repeated use of single site of action herbicides in short term crop-rotations with large quantities of winter cereals. The accumulation of NTSR loci in single plants increases the risk of biotypes with broad resistances against many different modes of action. Resistance linkages were found to be plant-specific which may result in unpredictable resistance situations in the field. Even pre-emergence herbicides can be affected by NTSR. An option to detect these resistances is provided by a soil-based greenhouse bioassay with pre-germinated seeds.
Exploring the effects of different stubble tillage practices and glyphosate application combined with the new soil residual herbicide cinmethylin against Alopecurus myosuroides Huds. in winter wheat
(2022) Messelhäuser, Miriam Hannah; Saile, Marcus; Sievernich, Bernd; Gerhards, Roland
Effective control of Alopecurus myosuroides Huds. (blackgrass) solely with a chemical treatment is not guaranteed anymore because populations exhibit resistance to almost all herbicide modes of action. Integrated weed management (IWM) against blackgrass is necessary to maintain high weed control efficacies in winter cereals. Four field experiments were conducted in Southwest Germany from 2018 to 2020 to control A. myosuroides with a combination of cultural and chemical methods. Stubble treatments, including flat, deep and inversion soil tillage; false seedbed preparation and glyphosate use, were combined with the application of the new pre-emergence herbicide cinmethylin in two rates in winter wheat. Average densities of A. myosuroides in the untreated control plots were up to 505 plants m−2. The combination of different stubble management strategies and the pre-emergence herbicide cinmethylin controlled 86–97% of A. myosuroides plants at the low rate and 95–100% at the high rate until 120 days after sowing. The different stubble tillage practices varied in their efficacy between trials and years. Most effective and consistent were pre-sowing glyphosate application on the stubble and stale seedbed preparation with a disc harrow. Stubble treatments increased winter wheat density in the first year but had no effect on crop density in the second year. Pre-emergence application of cinmethylin did not reduce winter wheat densities. Multiple tactics of weed control, including stubble treatments and pre-emergence application of cinmethylin, provided higher and more consistent control of A. myosuroides. Integration of cultural weed management could prevent the herbicide resistance development.
Incorporating agronomic measures into integrated weed management strategies using pre-emergence herbicide cinmethylin to control Alopecurus myosuroides Huds.
(2022) Messelhäuser, Miriam; Gerhards, Roland
Alopecurus myosuroides Huds. is one of the most problematic grass weeds in cereal production in Western Europe. This grass weed spread rapidly due to the repeated and intensive use of herbicides with the same mode of action and changes in arable cropping and tillage systems. Herbicide applications are the common agricultural practice for successful control of A. myosuroides due to its high flexibility and low cost. However, due to European and national restrictions and the growth of herbicide-resistant populations, farmers are forced to reduce herbicide use to minimize chemical impacts on the environment and food chain. As a holistic approach for reducing herbicide use, integrated weed management (IWM) is a diversification of the control strategy of A. myosuroides. In this thesis, several aspects of IWM were examined and combined to test for a successful A. myosuroides control strategy in winter cereals. Special attention was paid to cinmethylin, a pre-emergence herbicide with a new mode of action in winter cereals to control A. myosuroides. The first article comprised the development of an agar bioassay sensitivity test to determine sensitivity differences in A. myosuroides populations to pre-emergence herbicides containing flufenacet and the re-discovered substance cinmethylin. All of the tested populations did not show reduced sensitivity to cinmethylin, but differences in resistance factors were observed between the agar bioassay sensitivity test and the standard whole plant pot bioassay in the greenhouse. Nevertheless, it was possible for the most part to confirm the results for cinmethylin and flufenacet of the standardized greenhouse whole plant pot bioassay in the agar bioassay sensitivity tests and hence create a reliable, faster test system. The second article focused on cultural measures like cover crop mixtures, various stubble tillage methods and glyphosate treatments and their effect on total weed infestation in particular on A. mysouroides and volunteer wheat. Within two field experiments, the cover crop mixtures and the dual glyphosate application achieved a control efficacy of A. myosuroides of up to 100%, whereas stubble tillage and the single glyphosate treatment did not reduce A. myosuroides population significantly. The results demonstrated, that besides a double glyphosate application, well developed cover crop mixtures have a great ability for weed control, even for A. myosuroides. The third article also dealed with the combination of cultural measures (delayed seeding) and herbicide application and their influence on A. myosuroides control efficacy and yield response of winter wheat and triticale. Results indicate that cultural methods such as delayed seeding can reduce A. myosuroides populations up to 75%, although to achieve control efficacy of > 95%, supplementary herbicides should be used. In the fourth article, a two-year experiment on two experimental sites was set up with a special focus on stubble tillage methods, glyphosate application and the application of the pre-emergence herbicide cinmethylin in two rates. Control efficiencies of 99-100% were achieved by ploughing, double glyphosate application or via false seedbed preparation, each in combination with a cinmethylin application. In the last article, over a period of three years the new pre-emergence herbicide cinmethylin was tested in combination with stubble treatments and delayed drilling of winter annual cereals in winter wheat and winter triticale in Southwestern Germany. Cinmethylin controlled 58-99% of A. myosuroides plants until 120 days after sowing. Additive and synergistic effects of cinmethylin and delayed drilling were found for all studies. In this study, the focus was set on monitoring, cultural and direct weed control methods. Considering especially A. myosuroides, a diverse control strategy needs to be implemented to ensure a sustainable and reduced herbicide use, high control levels, minimized crop damage, safeguarded grain yields and reduced risk of resistance development. However, IWM measures imply increased system complexity, which may make their adoption by farmers difficult. Nevertheless, the results show that cinmethylin can be successfully used for weed control systems in combination with different stubble tillage methods, glyphosate application, delayed seeding, or herbicide sequences and mixtures, making it a valuable tool in integrated weed and resistance management strategies with its novel and unique mode of action.
Integrated management, analysis of mechanisms and early detection of resistant populations of Alopecurus myosuroides HUDS. and Apera spica-venti L. Beauv.
(2015) Kaiser, Yasmin; Gerhards, Roland
The control of pests is one of the major challenges in agricultural production worldwide. Especially weeds cause severe yield losses by competing with crops for light, space, water and nutrients. Due to the relatively low costs for acquisition and application of herbicides and a high control efficacy, chemical measures are predominantly applied to control weeds. In Europe, Alopecurus myosuroides HUDS. (blackgrass) and Apera spica-venti L. Beauv. (silky windgrass) are major weeds especially in winter wheat. The occurrence at high population densities in combination with a consequent use of herbicides with the same modes of action has resulted in the selection of resistant populations. Populations with target-site resistance (TSR) as well as non-target-site resistance (NTSR) could be confirmed for A. myosuroides and A. spica-venti. In contrast to the mechanisms of TSR, NTSR mechanisms are less investigated. Due to the steadily increasing number of putative herbicide resistant weed populations, the demand for rapid resistance tests is rising. The papers of the dissertation focus on the integrated management, the investigation of resistance mechanisms and the detection of herbicide resistant weed populations. The following research objectives have been examined within the four work packages (papers): – To develop a new methodology for a rapid detection of herbicide resistance and to confirm that results are comparable with classical greenhouse approaches – To investigate metabolism of herbicides in sensitive and resistant populations of A. myosuroides to gain comprehensive knowledge on resistance mechanisms – To evaluate the influence of agronomic factors on the probability of resistance occurrence and to develop a geo-referenced database for mapping the spread of herbicide-resistant A. spica-venti populations across Europe – To assess the influence of crop rotation and herbicide strategies on population development and herbicide resistance of A. myosuroides and crop yield The four papers come to the following results regarding the main research objectives: 1st paper: A laboratory test was developed to accelerate the detection of herbicide resistance. Therefore, A. myosuroides was cultivated in wellplates containing nutrient agar and herbicides. The evaluation of herbicide resistance was conducted by a sensor, measuring chlorophyll fluorescence. The results of the developed test corresponded well to the standard whole-plant pot tests in the greenhouse. In both tests sensitive and resistant populations were identified, however results of the Chlorophyll Fluorescence Imaging were available earlier. 2nd paper: Metabolism of herbicides was investigated in populations of A. myosuroides by using liquid chromatography - tandem mass spectrometry (LC-MS/MS) to gain comprehensive knowledge on mechanisms of herbicide resistance. NTSR populations differed from sensitive and TSR A. myosuroides in form of an enhanced degradation of the active ingredient or metabolite, depending on the investigated herbicide. For the investigated herbicides (inhibition of ACCase and ALS) it was shown that herbicide metabolism plays an important role regarding herbicide resistance in A. myosuroides. 3rd paper: To evaluate the influence of agronomic factors on the probability of resistance occurrence in A. spica-venti, numerous populations were screened in the greenhouse. The corresponding field history obtained from questionnaires and the results of greenhouse assays were used to develop a GIS-database in which herbicide-resistant A. spica-venti populations were mapped. The statistical analysis revealed that a high percentage of winter crops in the crop rotation, together with conservation tillage, early sowing dates and high population density increased the occurrence of herbicide resistance in A. spica-venti. 4th paper: To assess the impact of crop rotation and herbicide strategies on A. myosuroides, field studies at two locations in Southern Germany have been carried out. Results show that densities of A. myosuroides increased in continuous winter wheat. The introduction of spring crops significantly reduced densities, even without using herbicides. Furthermore it has been shown that the risk of herbicide resistance was reduced when performing a consequent change of herbicide mode of action. The use of herbicides with only one mode of action increased the number of herbicide resistant plants. Crop yield was notably influenced by A. myosuroides in winter wheat. The overall results of this dissertation showed the great impact of agricultural measures on herbicide resistance in A. myosuroides and A. spica-venti and demonstrated opportunities for prevention and management. The developed resistance quick test provides an accelerated detection of herbicide resistance and therefore the chance to initiate resistance management strategies much earlier.
Investigations on herbicide resistance in Apera spica-venti populations
(2011) Massa, Dario; Gerhards, Roland
Weeds belong to the main biotic yield-limiting factors in agricultural fields worldwide. Since the introduction of herbicides in the global market more than six decades ago, agro-ecosystems have been characterized by a strong reliance on herbicides for weed management. However, the selection pressure exerted on weed populations by long-term application of herbicides with the same mode of action has imposed selection for resistance within several weed species. Apera-spica-venti (L.) Beauv. represents one of the most abundant annual grass weeds in autumn-sown crops of Central and Eastern European arable lands. Since the first report on herbicide-resistant A. spica-venti populations in 1994, several cases have been documented. Therefore, it is assumed that this species is evolving resistance to herbicides. The main objectives of the present work were: 1. The development of reliable testing procedures under greenhouse conditions for the verification of herbicide resistance in A. spica-venti populations; 2. The quantification of resistance in A. spica-venti at different herbicide doses using dose-response assays and digital image analysis; 3. The elucidation of resistance mechanisms in A. spica-venti populations at the molecular genetic level through laboratory experiments; 4. The evaluation of the influence of farm management factors on the occurrence and spread of herbicide resistance in A. spica-venti populations through the use of statistical modeling; 5. The development of a geo-referenced database for documenting the spatial and temporal distribution of herbicide-resistant A. spica-venti populations in Europe; 6. The verification of the results obtained from the greenhouse assays under realistic field conditions and the detection of the introgression of herbicide resistance traits into the sensitive population after two generations. The main resistance testing procedure consisted of growing plants in the greenhouse from seeds collected in the suspect fields and spraying them with herbicides. Applications were carried out with an automated precision sprayer either at a single dose or at a range of doses. Herbicide efficacy was then assessed at 15 and 30 days after treatment by direct comparison with untreated controls. Results from the greenhouse assays showed that most of the screened popula¬tions (~70%) have evolved resistance to herbicides, particularly acetolactate synthase (ALS)-inhibitors. Dose-response assays revealed resistance factors at the ED90 (ED90 resistant / ED90 sensitive) of up to 140 after treatment with flupyrsulfuron-methyl, thus indicating that A. spica-venti is a resistance-prone grass weed. A novel method for quantifying resistance based on the assessment of percent canopy cover using digital image analysis has been developed in the greenhouse to provide a potential alternative to the labour-intensive and time-consuming dry weight assessments. Laboratory experiments conducted on over 70 ALS-resistant populations revealed that target site mutations were responsible for the observed resistance. Cleaved Amplified Polymorphic Sequences (CAPS) marker analysis and sequencing of the amplified 3? and 5? ends of the ALS gene by Rapid Amplification of cDNA Ends (RACE) revealed the presence of previously known as well as novel mutations endowing resis¬tance to ALS-inhibitors. The development of a risk assessment model allowed the evaluation of the influence of farm management factors on the probability of resistance occurrence in A. spica-venti populations. Results showed that a high percentage of winter crops in the rotation (>75%), together with reduced or no-till practices, early sowing and high population density significantly increase the risk of resistance emergence. An internet-based geo-referenced database was developed to document the spatial and temporal distribution of herbicide-resistant A. spica-venti populations in Europe. Finally, field experiments were carried out over the course of two years (2008/2010) by sow-ing ALS-resistant and susceptible A. spica-venti populations in winter wheat plots. Resistant plants survived herbicide application, completed their life cycle and set vital seeds, which showed a significantly higher germination rate compared to the sensitive population. However, maximum yield losses of only ~10% could be ob¬served at A. spica-venti panicle densities of >400 m-2. Greenhouse bioassays conducted at the end of the first year with plants from seeds collected in the sensitive plots showed an increase in tolerance to ALS-inhibitors of ~20%, thus suggesting introgression of herbicide resis¬tance traits into the sensitive population already after one year. The outcomes of this work add knowledge to the understanding of the mechanisms underlying resistance to herbicides in A. spica-venti populations and provide weed scientists and consultants with useful tools for the reliable diagnosis and prevention of herbicide resistance in weed populations.
Investigations on herbicide resistant grass weeds
(2009) Balgheim, Natalie; Balgheim, Natalie
Weeds are one of the most troublesome threats for farmers, causing high yield losses and serving as hosts for pathogens and insect pests. Since the introduction of chemical weed control agricultural production systems have changed. During the last years the number of herbicide resistant grass weeds is steadily increasing especially in cereal monocultures. These monocultures are characterised by the repeated use of herbicides with the same modes of action and minimum-tillage practices. All these factors can one by one or all together lead to the development of herbicide resistant grass weeds. In general herbicide resistance is the result of heritable changes to biochemical processes that enable plant survival when treated with herbicides. Two different mechanisms are commonly known to confer resistance: target-site resistance and non-target-site resistance. First is the result of an altered target enzyme, where a single point mutation is changing the amino acid structure and exclude herbicide from effectively binding to the target enzyme. The second one, non-target-site resistance, can be summarised as the mechanisms which includes all other mechanisms than target-site resistance, for example rapid metabolic degradation or translocation of herbicides. In Germany, the most trouble causing weeds associated with target-site resistance are the grass weeds Alopecurus myosuroides Huds. and A. spica-venti L. Beauv.. All investigations carried out during this thesis are dealing with those two weed species. Therefore the main objectives of this thesis are the following: To characterise the resistance levels and patterns of both species. To identify the underlying resistance mechanisms. To develop molecular markers for rapid detection of target-site based resistance. To get an idea of the spatial and temporal distribution of herbicide resistant grass weeds in arable fields. Both investigated species are highly adapted to cereals and developed resistance against ACCase and ALS inhibiting herbicides. So they are an increasing problem for German farmers and in consideration of the fact, that both weeds have developed multiple resistances, detecting and management strategies for controlling and preventing of these weeds are absolutely necessary. Carried out dose response relationships proved strong resistance of the A. myosuroides biotype BR(R) against cycloxydim and fenoxaprop, where low resistance was expressed against clethodim. However, biotype BL(R) showed resistance to fenoxaprop and clodinafop only. Dose response experiments carried out with the A. spica-venti biotype showed resistance to sulfosulfuron and iodo-/mesosulfuron. No cross resistances could be detected in both species. The carried out DNA analysis revealed target-site resistance as the underlying resistance mechanism. BR(R) and BL(R) showed well known substitutions: an amino acid change on position 1781 with in the CT domain result in a change of Leu to Ile which confers resistance to APPs and CHDs in the biotype BR(R). The mutation of Gly to Ala on position 2096 within the CT domain causes resistance to APPs only. Also in the A. spica-venti biotype a amino acid change is the responsible resistance mechanism: a change of Pro to Thr at position 197. These sequencing results serve as basis for the development molecular markers. Designed markers based on dCAPS technology. Such markers were developed to detect SNPs which can cause amino acid changes on the constitutive enzymes. Developed markers can rather differentiate between heterozygous and homozygous resistant alleles. Their technology is based on the fact that restriction endonucleases can cut DNA strands on specific recognition sites. This fact can be used for developing markers which are cutting the DNA in a previously generated PCR fragment on the mutation or wild-type sites, respectively. If there is no recognition site, it can be implemented by specific primers during the PCR. By these markers suspicious samples can be analysed and the results give an advice for management strategies, because target- and non-target-site resistance need different controlling strategies. Investigations on the spatial and temporal distribution of weed populations where carried out on an arable field, invested with herbicide resistant A. myosuroides. Collected and analysed leave samples give information about the spatial dynamics of homozygous, heterozygous and sensitive plants in the field. Results show that the distribution of resistant plants depends on the weed density. Besides the weeds are distributed heterogeneous on the field and occur in patches that are persistent over several years. This example revealed that herbicide resistance is rather associated with crop cultivation measurements. Changes in herbicidal and cultivation measurements shall be practiced to control and to prevent the occurrence of herbicide resistant grass weeds.
Population genomics of herbicide resistance in Alopecurus myosuroides
(2022) Kersten, Sonja; Schmid, Karl J.
Over the past 50 years, herbicides have often replaced mechanical and manual human weed control, thus representing a major factor in yield productivity in modern agriculture. Herbicide applications, however, exert strong selection pressures on weeds. As a consequence, these species have developed herbicide resistance through adaptive, beneficial alleles that increase in number to ensure the persistence of the populations, a phenomenon known as evolutionary rescue. A major research question is whether herbicide resistance adaptation is more likely to arise from standing genetic variation that was present before the onset of herbicide selection or from de novo mutations that arose after herbicide selection began. To address this question, I focused on target-site resistance (TSR) point mutations, which cause a lower binding affinity to the target protein of the respective herbicides. I first investigated the diversity of TSR haplotypes in populations of the grass species Alopecurus myosuroides (common name: blackgrass), and compared it with the TSR diversity outcome of simulated populations under both evolutionary scenarios. I first conducted a population genetics study of A. myosuroides, which is the most problematic weed in winter cereals across the European continent due to rapid resistance evolution. To obtain genome-wide polymorphic markers, I adapted a restriction site-associated DNA sequencing protocol to this species. I began by analyzing the diversity and population structure in a smaller local South German collection. The fact that I could differentiate populations on a local scale motivated me to extend the study to a European-wide collection, in which I found clear population structure, albeit with low differentiation and some evidence for admixture across Europe. In addition, I generated highly accurate long-read amplicons from single individuals of two loci, ACETYL-COA CARBOXYLASE (ACCase) and ACETOLACTATE SYNTHASE (ALS), which are the targets of the two main herbicide modes of action used in European cereal crops. I obtained completely phased haplotype information, supporting the analysis of haplotype diversity on a population level. I found a remarkable diversity of beneficial TSR mutations at the field level arising from multiple haplotypes of independent origin, so called soft sweeps. I used this information to perform forward simulations to investigate the evolutionary origin of these mutations. I found evidence that a majority of resistance mutations originated from standing genetic variation. While this at first may appear surprising, it is consistent with very large census and effective population sizes in blackgrass. Since long-read amplicon sequencing of single individuals could be costly and time consuming, I extended the analysis to pools of 150 to 200 individuals from Germany, Belgium, France, the Netherlands and the United Kingdom. By combining the power of a more stringent accuracy criterion in our long-reads and a novel clustering software (PacBio amplicon analysis), I was able to preserve individual haplotype information in pooled samples. Furthermore, in a proof of concept experiment, I was able to recover in our pools most haplotypes previously sequenced in individuals. The amplicon study provides a versatile workflow that can be easily adapted to any gene of interest in different species. In conclusion, I found that many A. myosuroides populations likely already have the genetic prerequisites not only for rapid evolution of resistance to currently used herbicides, but also to herbicides that have not yet been brought to market.