Institut für Phytomedizin

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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.
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.

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