Browsing by Subject "Herbizid"

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Adaptation of model organisms and environmental bacilli to glyphosate gives insight to species-specific peculiarities of the shikimate pathway
(2024) Schwedt, Inge; Commichau, Fabian M.
Glyphosate (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.
An image analysis and classification system for automatic weed species identification in different crops for precision weed management
(2010) Weis, Martin; Gerhards, Roland
A system for the automatic weed detection in arable fields was developed in this thesis. With the resulting maps, weeds in fields can be controlled on a sub-field level, according to their abundance. The system contributes to the emerging field of Precision Farming technologies. Precision Farming technologies have been developed during the last two decades to refine the agricultural management practise. The goal of Precision Farming is to vary treatments within fields, according to the local situation. These techniques lead to an optimisation of the management practice, thereby saving resources, increasing the farmers outcome, reducing the overall management costs and the environmental impact. A successful introduction of Precision Farming involves the development of application equipment capable of varying treatments and sensor technology to measure the spatial heterogeneity of important growth factors. Such systems are able to record, store and use large amounts of data gathered by the sensors. Decision components are needed to transform the measurements into practical management decisions. Since the treatments are varied spatially, positional data, usually measured using GPS technology, has to be processed. The located measurements lead to a delineation of management zones within a field and are represented by geo-data and can be visualised in maps. The improved, detailed knowledge of the situation within the field leads to new and extended scopes of applications and allows to document the management practices more precisely. In this work, parts of Precision Farming technology were developed for site-specific weed management. Five selected publications are presented, covering the technological prerequisites and details of the developed system.
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.
Effects of weeds on yield and determination of economic thresholds for site-specific weed control using sensor technology
(2014) Keller, Martina; Gerhards, Roland
Weeds can cause high yield losses. Knowledge about the weeds occurring, their distribution within fields and their effects on the crop yield is important to achieve effective weed control. The critical period for weed control (CPWC) and the economic threshold (ET) are important key concepts and management tools in weed control. While the former helps to time weed control in crops of low competitiveness, the latter provides a decision aid to determine whether weed control is necessary. This decision is generally taken at the field level. Weeds have been found to be distributed heterogeneously within fields. Site-specific weed control (SSWC) addresses this sub-field variation by determining weed distribution as input, by taking control decisions in the decision component and by providing control measures as output at high spatial resolution. Sensor systems for automated weed recognition were identified as prerequisite for SSWC since costs for scouting are too high. While experiences with SSWC using sensor data as input are still scarce, studies showed that considerable herbicide savings could be achieved with SSWC. ETs can serve as thresholds for the decision component in SSWC systems. However, the commonly used ETs were suggested decades ago and have not been updated to changing conditions since. The same is the case for the CPWC in maize in Germany. In addition, the approaches to determine the CPWC are usually not based on economic considerations, which are highly relevant to farmers. Thus, the objectives of this thesis are: 1. To test different models and to provide a straightforward approach to integrate economical aspects in the concept of the CPWC for two weed control strategies: Herbicide based (Germany) and hoeing based (Benin); 2. To determine the effect of weeds on yield and to calculate ETs under current conditions which can be used for SSWC; 3. To evaluate the use of bi-spectral cameras and shape-based classification algorithms for weed detection in SSWC; and 4. To determine changes in weed frequencies, herbicide use and yield over the last 20 years in southwestern Germany. Datasets in maize from Germany and Benin served as input for the CPWC analyses. The log-logistic model was found to provide a similar fit as the commonly used models but its parameters are biologically meaningful. For Germany, analyses using a full cost model revealed that farmers should aim at applying herbicides early before the 4-leaf stage of maize. In Benin, where weed control is mainly done by hoeing, analyses showed that one well- timed weeding operation around the 10-leaf stage could already be cost-effective. A second weeding operation at a later stage would assure profit. The precision experimental design (PED) was employed to determine the effect of weeds, soil properties and herbicides on crop yield in three winter wheat trials. In this design, large field trials’ geodata of weed distribution, herbicide application, soil properties and yield are used to model the effects of the former three on yield. Galium aparine, other broadleaved weeds and Alopecurus myosuroides reduced yield by 17.5, 1.2 and 12.4 kg ha-1 plant-1 m2 determined by weed counts. The determined thresholds for SSWC with independently applied herbicides were 4, 48 and 12 plants m-2, respectively. Bi-spectral camera based weed–yield estimates were difficult to interpret showing that this technology still needs to be improved. However, large weed patches were correctly identified. ETs derived of field trials’ data carried out at several sites over 13 years in the framework of the ’Gemeinschaftsversuche Baden-Württemberg’ were 9.2-9.8 and 4.5-8.9 % absolute weed coverage for winter wheat and winter barley and 3.7% to 5.5% relative weed coverage for maize. Overall, the weed frequencies in winter cereals were found to be more stable than the weed frequencies in maize during the observation period. In maize, a frequency increase of thermophilic species was found. Trends of considerable yield increases of 0.16, 0.08 and 0.2 t ha-1 for winter wheat, winter barely and maize, respectively, were estimated if weeds were successfully controlled. In order to evaluate the use of bi-spectral cameras and shapebased classification algorithms for weed detection in SSWC, herbicides were applied site-specifically using weed densities determined by bi-spectral camera technology in a winter wheat and maize field. Threshold values were employed for decision taking. Using this approach herbicide savings between 58 and 83 % could be achieved. Such reductions in herbicide use would meet the demand of society to minimize the release of plant protection products in the environment. Misclassification occurred if weeds overlapped with crop plants and crop leaf tips were frequently misclassified as grass weeds. Improvements in equipment, especially between the interfaces of camera, classification algorithms, decision component and sprayer are advisable for further trials. In conclusion, the derived ETs can be easily implemented in a straightforward SSWC system or can serve as decision aid for farmers in winter wheat and winter barley. Further model testing and adjusting would be necessary. For maize, the use of ETs at the field level is not suggested by this study, however the need for early weed control is clearly demonstrated. Bi-spectral camera technology combined with classification algorithms to detect weeds is promising for research use and for SSWC, but still requires some technical improvements.
From greenhouse to field practice : herbicide resistance detection using chlorophyll-fluorescence-imaging technology
(2017) Wang, Pei; Gerhards, Roland
All over the world, herbicide resistance has developed to one of the most important barriers in weed control, making the implementation of the weed control strategy more complicated. There is an intense need for a rapid, cheap and reliable method to conduct in field detection of herbicide resistant weed populations. In the current thesis with the use of chlorophyll fluorescence imaging technology, such a method is implemented and tested in field conditions. A series of experiments were designed and carried out. The data gathered from these experiments were compiled under three paper articles. Paper 1. A greenhouse experiment was conducted to verify if the parameter, Maximal Photosystem II Quantum Yield (Fv/Fm), could possibly indicate the herbicide efficacy. The chlorophyll-fluorescence-imaging sensor, Weed PAM®, was selected for the measurements. In the first part it was investigated if the Fv/Fm value could differentiate between herbicide sensitive and resistant plants. In the second part two important abiotic stress factors were tested if they affected the Fv/Fm value. I) Six herbicides were tested on herbicide sensitive and resistant Alopecurus myosuroides populations; II) Water shortage and nitrogen deficiency were applied on a herbicide sensitive population to observe their influence on the plants. The sensitive plants presented significantly lower Fv/Fm values than the resistant plants 3 days after treatment (DAT) for the ALS and ACCase inhibitors. On the same day, and for the same treatments the Fv/Fm values of the resistant plants were not affected and similar to the control. Appling a PS II inhibitor reduced the Fv/Fm values of both sensitive and resistant plants rapidly. Yet, sensitive and resistant plants could clearly be separated on 4 DAT based on the different Fv/Fm values. On the other hand, nitrogen deficiency did not influence the photosystem II measurements. Water shortage reduced rapidly the Fv/Fm value of the plants seven days after the application, yet at this point plant symptoms included the death of the plants. According to this experiment, the Weed PAM® sensor has proved its capability to identify the sensitive and resistant A. myosuroides populations shortly after the herbicide application. Paper 2. A verification of the above results was made under field conditions for different A. myosuroides populations and different locations. On the first part 50 populations in total including both sensitive and herbicide resistant populations were tested. The second part field experiments were conducted in ten locations around Germany over two years with the local field population mix. It was investigated if the Weed PAM® sensor could separate between herbicide sensitive and resistant A. myosuroides populations 5 DAT. The different populations were sown in a winter wheat field. Two ACCase- and three ALS- inhibitors were applied. In all herbicide treatments, Fv/Fm values of A. myosuroides were significantly lower than the untreated plants at the 5 DAT. For each location, measurements were conducted at 5 DAT. A visual measurement, to verify the result, was carried out at 21 DAT. In both cases, 95% of the plants were correctly identified as sensitive or resistant. This demonstrated the ability of the Weed PAM® sensor to conduct in field real time detection of herbicide resistant A. myosuroides populations shortly after treatment. Paper 3. Greenhouse and field experiments were carried out to investigate if the chlorophyll fluorescence of soybean plants was altered, under herbicide stress. Herbicide combinations including inhibitors of PS II, DOXP synthase, cell division and microtubule assembly were selected for different pre-emergence treatments. Herbicide combinations including inhibitors of PS II, ALS and ACCase were applied in post-emergence treatments. Chlorophyll fluorescence was measured from the emergence of soybeans until the three/four-leaf stage. Furthermore the stress effect of the different treatments on the soybean plants was determined by measuring their dry biomass. In the greenhouse, post-emergence treatments with ALS and ACCase inhibitors did not seem to induce stress on the soybean plants. As expected, it originally demonstrated low Fv/Fm values when stressed by PS II inhibitors. But the PS II system recovered soon, one week after emergence. Stress induced by other pre-emergence herbicides occurred one week after emergence and lasted longer than the stress induced by the PS II inhibitors. Dry biomass collaborated with the sensor result. Based on the current thesis, the Weed PAM® system can be an important tool in the identification of herbicide resistant weed populations, in a timely manner. It has proven its capabilities both in A. myosuroides as a weed and in soybean plants. This technology will help farmers to take more suitable weed control strategies, as well as less economic and environmental risks.
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.
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.
Studies on the efficacy, composition and mode of action of an ethoxylated soybean oil adjuvant for herbicides
(2012) Heini, Julia; Gerhards, Roland
The potential of an ethoxylated soybean oil adjuvant ? Agnique® SBO 10 ? to increase the efficacy of different herbicides was investigated in the present thesis. Furthermore, Agnique® SBO 10 was fractioned by preparative High-Performance Liquid Chromatography (prep. HPLC) to elucidate the composition of the ethoxylated soybean oil (ESBO). In addition, experiments with fluorescein-labelled Agnique® SBO 10 were conducted to evaluate the fate of modified seed oil adjuvants on the leaf surface. The efficacy of many herbicides can be increased by adding adjuvants to the spray solution. Adjuvants, in particular surfactants, are able to increase the foliar uptake of active ingredients for example, by enhancing the retention of spray droplets on cuticles, penetration and absorption into leaf tissue. Agnique® SBO 10 belongs to a group of environmental friendly surfactant containing ten ethylene oxide units. Modified seed or vegetable oils are biodegradable and are proposed to be as effective as petroleum oils. These facts make them very interesting for the usage as adjuvants for agrochemicals. To evaluate the potency of Agnique® SBO 10 dose-response studies were accomplished. Results showed that sulfosulfuron, topramezone, and foramsulfuron & iodosulfuron did not control velvetleaf (Abutilon theophrasti Medik.) sufficiently when they were applied without adjuvant. Agnique® SBO 10 acted 2-fold better compared to the recommended adjuvants. Thus, Agnique® SBO 10 could present an alternative adjuvant for a widespread use. These results show, that a certain adjuvant has the potential to increase the efficacy of a herbicide to its maximum. However, for the user it is not easy to choose a proper adjuvant of the broad range of available products. Thus, integrating additives into pesticide formulations is desirable. Due to the fact, that Agnique® SBO 10 is a huge and complex product it cannot be included into a formulation. For this reason, an experiment was accomplished dividing Agnique® SBO 10 into four fractions by using preparative HPLC. These four fractions were isolated of Agnique® SBO 10 and tested in combination with commercially formulated herbicides (foramsulfuron & iodosulfuron and bromoxynil octanoate) and a technical grade bromoxynil product. The aim was to find a fraction with a higher potential compared to Agnique® SBO 10 to be used as low-molecular adjuvant. The herbicide-fraction mixtures were applied as droplets with a pipette on velvetleaf leaves. Dry weight and leaf area measurements of test plants demonstrated that the efficacy of foramsulfuron & iodosulfuron and the technical bromoxynil was increased with decreasing polarity of isolated fractions of Agnique® SBO 10, whereas the efficiency of bromoxynil octanoate was tendentially enhanced adding more polar fractions to the herbicide solution. Again, it was demonstrated that the herbicide?s efficacy is strongly dependent of the adjuvant/fractions. Though a lot of research was conducted to confirm the positive effect of adjuvants on herbicides, their mode of action is still not completely clear. Because modified vegetable oils are complex compounds, a radioactive labelling is not easy. Therefore, in this study Agnique® SBO 10 was labelled with fluorescein to investigate, whether Agnique® SBO 10 remains on the leaf surface, accumulates in the cuticle or even penetrates into the underlying plant tissue. Fluorescein-labelled Agnique® SBO 10 (AF) was applied to one leaf of the test species velvetleaf, wild mustard and sugar beet. At different time intervals, treated leaves were harvested and rinsed with different washing solutions (deionised water, methanol/water and chloroform). The washed leaves and the respective washing solutions were processed separately and analyzed with HPLC coupled with an UV detector. Results lead to the assumption that AF might have been passed through the cuticle. However, AF is not expected to be very mobile because it is lipophilic and electrically neutral. If there was a penetration through the cuticle, an enzymatic metabolisation of Agnique® SBO 10 into fatty acids and ethoxylated glycerol is likely to occur. With this thesis the herbicide-enhancing effect of Agnique® SBO 10 was confirmed. Furthermore, a new approach for the design of optimized adjuvants for precise herbicide-adjuvant mixtures was presented. Though a lot of research has to be conducted to elucidate the mode of action of adjuvants, this study gives an approach to investigate the behaviour of foliar applied adjuvants.
The biocontrol agent Fusarium oxysporum f. sp. strigae - Monitoring its environmental fate and impact on indigenous fungal communities in the rhizosphere of maize
(2016) Zimmermann, Judith; Cadisch, Georg
The fungal biocontrol agent (BCA) Fusarium oxysporum f. sp. strigae (Fos) has proven to be effective in the suppression of the parasitic weed Striga hermonthica, which causes substantial yield losses in cereals in Sub-Saharan Africa. A prerequisite for widespread implementation of the biocontrol technology is the official registration of the BCA Fos by country authorities in Sub-Saharan Africa. The FAO and OECD institutions established international registration regulations to ensure the environmental safety of microbial BCAs. The present thesis aimed on assessing the potential of the BCA Fos to meet these registration requirements and was, therefore, based on the following two major objectives: (1) A specific DNA-based monitoring tool for Fos was developed which allows following its population kinetics in soils as driven by contrasting environmental impacts, such as soil type, plant growth stage and seasonality. (2) Risk assessment studies were conducted to assess potential side effects of Fos inoculation on non-target soil microorganisms.
Untersuchung alternativer Unkrautmanagementsysteme für Kulturraps unter Einbeziehung von Möglichkeiten zur Reduzierung des Auftretens von Raps als Durchwuchs
(2022) Schwabe, Sebastian; Claupein, Wilhelm
Oilseed rape is the worlds second most important oil crop after soybeans. In the course of the European Green Deal of the European Union and the associated stronger promotion of renewable energies in the future, it can be assumed that the importance of oilseed rape cultivation will continue to increase. Due to the price pressure on conventional farms in Germany, cultivation systems have been changed from an economic point of view, partly to the detriment of sustainability. As a re-sult, crop rotations often consist of a few monetarily profitable crops and the proportion of spring crops in the crop rotation is reduced. Oilseed rape cultivation is attractive from an economic point of view, and its share in the crop rotation has been increased. The intensity of tillage and mechanical weed control has been reduced. The weed control success is strongly dependent on the effectiveness of numerically limited herbicidal active agents. Due to monotonous crop rotations and the lower tillage intensity, certain weed species are pro-moted more strongly, while at the same time these are controlled with only a few herbicid-al active agents. Adapted, difficult-to-control, and in some cases herbicide-resistant weed populations develop. For this reason, the purpose of this thesis is to evaluate alternative weed management systems in oilseed rape, while also investigating options for prophylac-tic prevention of the emergence of volunteer oilseed rape as a weed in crop rotation. The objectives of this thesis were: (i) To evaluate the feasibility of hoeing as a mechanical weed control method and the application of the Clearfield® system in oilseed rape as a comparison to common, field herbicide strategies. (ii) To focus on the volunteer oilseed rape issue. Volunteers resulting from Clearfield® oilseed rape are more difficult to control chemically in subsequent crops due to inherited herbicide tolerance. The potential of differ-ent seed treatments in oilseed rape to reduce the development of secondary dormancy, and therefore seed persistence in the soil and the volunteer oilseed rape issue, was investigat-ed. Following these objectives, several field and laboratory experiments were conducted to generate data for three published scientific papers. Paper I: A two-year field trial was conducted to evaluate the performance of the Clearfield® system in oilseed rape under different management intensities compared to a more com-monly used pre-emergence herbicide system. The Clearfield® system is an alternative weed management system for oilseed rape. It is a combination of a broad-spectrum post-emergence herbicide and a Clearfield® oilseed rape variety that has tolerance to the herbi-cide. This tolerance was implemented in Clearfield® oilseed rape varieties through conven-tional, non-GM breeding techniques. Clearfield® herbicides have lethal effects on non-Clearfield® oilseed rape varieties. Paper II: An investigation was made through laboratory and field trials on the effect of ger-mination-promoting substances (nutrients and gibberellic acid) on the development of sec-ondary dormancy of oilseed rape seeds and on their persistence in the soil. Paper III: In a three-year field trial, hoeing as a weed control method was compared with a commonly used herbicide strategy. The hypotheses made in the introduction were both confirmed and refuted by the findings obtained in the trials. Hypotheses stated in paper I: (i) The Clearfield® herbicide and herbicides of a common practice pre-emergence strategy show similar efficiencies; (ii) Management intensity has an effect on weed density but does not affect yield; (iii) Herbicide strategy does not affect yield. At higher management intensities, both herbicide systems achieved comparable efficien-cies. At lower management intensities, especially in terms of seeding density and tillage, weeds were less efficiently controlled with the Clearfield®-system, and yields were par-tially lower. At higher management intensities, higher yields and lower weed emergence were observed compared to lower intensities, presumably due to better weed control by plowing and more favorable emergence conditions due to a higher tillage intensity. Hypotheses stated in paper II: (i) All tested substances reduce the induction of secondary dormancy; (ii) the tested sub-stances reduce the induction of secondary dormancy to different extents; (iii) the tested substances have an effect on the induction of secondary dormancy, regardless of whether the tested oilseed rape seeds originate from varieties with a high or low tendency, to devel-op secondary dormancy; (iv) if a variety tends to develop high secondary dormancy, its in-duction is reduced to a greater extent by the tested substances than in seeds from a variety with a low tendency to develop secondary dormancy. Most of the tested substances reduced both the induction of secondary dormancy and the survivability of oilseed rape seeds. The efficiency of the reduction depended on the type of substance and the oilseed rape variety. Substances containing gibberellic acid proved most effective, followed by micronutrient treatments and potassium nitrate. Hypotheses stated in paper III: (i) Hoeing achieves the same weed control efficiency as herbicides; (ii) regardless of whether herbicides or hoeing are used as weed control, the same oilseed rape yield can be realized. Weed biomass was higher compared to herbicide application when hoeing was used as a weed control measure. This is probably due to the weather-dependent efficiency of hoe-ing and its only partial surface applicability. Weeds emerging in or close to the seed row cannot be controlled. Nevertheless, no yield differences were found between hoeing as a weed control measure and pure chemical weed control. The competitive strength of the varieties used was most likely large enough to ensure this yield stability. Both hoeing and applying the Clearfield® system under higher management intensities re-sulted in similarly high oilseed rape yields compared to conventional herbicide strategies, although weed control efficiency was lower. As long as no hard-to-control weeds occur on a conventional farm and a common herbicide strategy effectively controls existing weeds, changing the weed management system is less beneficial. Because the Clearfield® system increases selection pressure on weeds, the occurrence of weed herbicide resistance be-comes more likely. In addition, Clearfield® volunteer canola is more difficult to control chemically in subsequent crops. Hoeing efficiency is weather dependent, weeds are only captured between rows, and area performance is lower. However, when difficult-to-control weeds increasingly minimize the efficiency of existing, conventional chemical control strategies, both hoeing and the use of the Clearfield® system in combination with a common herbicide strategy can expand weed control options and in-crease their efficiencies. When applying the Clearfield® system, strategies should be em-ployed to minimize the occurrence of Clearfield® volunteer oilseed rape in subsequent crops. It has been shown in this thesis that germination-promoting compounds, particularly gib-berellic acid, prevent the induction of secondary dormancy, as well as the ability of oilseed rape seeds to persist, and therefore, have the theoretical potential to contribute to a reduc-tion in the volunteer oilseed rape occurrence problem. Overall, both hoeing and the application of the Clearfield® system as alternative weed man-agement systems can usefully complement established methods of weed control in oilseed rape, where necessary. In addition, the use of germination-promoting compounds in oilseed rape seeds has demonstrated the theoretical potential to contribute to a reduction of volun-teer oilseed rape as a weed in crop rotations.