Browsing by Subject "Target-site 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.
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.