Summary
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On the 26th of September 2019, we visited the Bayer Forward Farm in Rommerskirchen
We visited the Bayer Forward Farm in Rommerskirchen, the “Damianshof” accompanied by Bernd Olligs (farmer and owner of the farm), Dr Patrick Beuters (Product Development Manager for fungicides in cereals, sugar beet and potatoes) and Karl Eschenbacher (Head of Bayer Forward Farming).
The farm in Rommerskirchen is one of two locations in Germany where Bayer established a Forward Farm: An experimental testing area for novel approaches to solve problems in the field of agriculture. Thereby, the farm’s purpose is also to educate the public about farming and emerging technologies. The engagement with the public and the promotion of face to face interaction is one of the foundations the project is based on.
During our visit, we received extensive first-hand insights into agriculture from different points of views. The different experts who accompanied us each represented a different important aspect of modern agriculture.
Dr Patrick Beuters
He is in the department of Market Development Manager for fungicides in cereals, sugar beet and potatoes.
Dr Patrick Beuters gave us insights into the importance of fungicides for agriculture. As a consultant for fungicides for cereals, sugar beet and potatoes he has an extensive overview about all their important products and modes of actions.
His explanations regarding the history and recent change in usage of fungicides as well as the latest interests in research completed the depictions of Karl Eschenbacher and Bernd Olligs very well.
At the same time, he stressed the need to investigate alternatives to meet future challenges. Furthermore, he showed us the extend of the recent interests of the industry for new alternatives, e.g. biologics.
According to Dr Beuters, it is important to work closely with farmers to ensure proper use of fungicides and effective food safety.
Karl Eschenbacher
He is Head of Bayer Forward Farming.
Karl Eschenbacher is the Supervisor Head of Bayer Forward Farming in Germany. He stressed the importance of a close cooperation between industry, farmer and consumer. A lack of understanding can easily lead to problems and misconceptions according to him.
He explained how valuable open communication about agricultural measures are to educate people about the necessity of certain methods and applications, e.g. the use of pesticides.
To reduce prejudices towards agriculture, engagement and education with the public are among the greatest tools to achieve a well-understood, fair-regulated, sustainable and safe food production.
Considering we live in a time, where technological advancements are achieved quickly and should be easily accessible and understood by the public, engagement with everyday people is often sparse. An open conversation about such topics and new approaches to communicate the subject in a target group specific manner can really make a difference.
To sum up, Karl Eschenbacher clarified how important well-informed consumers are to achieve an integrated agriculture.
Bernd Olligs
Is the farmer that is taking care of the farm Damianshof, where the forward farm is located
Bernd Olligs is the owner of the farm. He gave us great first-hand insight into the work as a farmer. He showed us how full of meaningful decisions his work is and underlined his statements with stories and anecdotes from his work.
His depictions of the life as a farmer made us realise how underestimated and unappreciated the work of farmers is in modern society and how complex the issues can be that they are confronted by. They have to assure a maximum yield while minimizing the costs and the impact on the environment while planning years far ahead. This is a task unmatched in modern society.
During our visit, Bernd Olligs also described the importance of improving the personal contact between farmers and consumers to create a broader understanding of the reasons that drive their decisions and our dependency on their experience.
General Experts
First Contact: 05.09
We approached Professor James Brown as an expert for fungal diseases of crops and other plants to receive an evaluation of our project as well as our concept for a potential application.
Prof. Brown gave us advice on various points of our project, which he depicted as an interesting approach that should quite certainly be a completely novel approach.
First of all, he portrayed that fungicides, although they can be unspecific and have off-target effects, are generally quite specific in their mode of action. Moreover, off-target effects are rather rare and usually weak or hard to detect. Besides this, we would have to put thought into how our system could have similar side effects, he explained. Also, he pointed out that specificity towards a certain pathogen, as well as a broader specificity can both be advantageous. For our system we would have to argue why it would be beneficial to have such a specific mode of action and off-target effects would be important enough to be worth avoiding.
Also, he pointed out, that the process of safety testing for fungicides is a rigorous, expensive and time-consuming one. During this process, the fungicides are extensively tested for potential negative impacts for the environment and for humans. This procedure takes nine to ten years after the discovery of the fungicide molecule. These tests are also an important reason, why fungicides generally have very few off-target effects if they are allowed to be commercially used. The patent life of fungicides lasts 17 years. Hence, the companies have only seven to eight years to significantly profit from the sales of a new fungicide while it is still in patent. This period can only be extended by patenting the manufacturing process or keeping important features of the product secret. The process for testing medical pharmaceuticals is quite analogous, although the period of safety testing and is two years shorter, he added. If our Troygenics would be aimed for a commercial use we should put more thought into product marketing and a strategy for IP protection to make sure to make a profit on the system, he suggested.
In his view, the short patent life of fungicides is also contributing to the problem of resistance because to make a quick return on their investment, companies have to sell as much of their product as fast as possible. The same effect applies to medical antibiotics, which results in them not being attractive investments for pharma companies anymore. For a commercial application a way to extend the patent life of the Troygenics would thereby be beneficial to maximise the potential profits.
If we knew of any possibility that fungi could evolve resistance to our Troygenics this would also have to influence our marketing strategy, Prof. Brown mentioned.
Regarding mathematic models he named us two further contacts to come in touch with.
One thing Prof. Brown especially pointed out, was that non-specific effects of fungicides are actually often advantageous and thereby desired by farmers because this substitutes multiple treatments with different pesticides. This way a crop can be sprayed just once and still be well protected from multiple diseases. Prof. Brown proposed, that the most important limitation for disease management on the fields is the number of opportunities over the course of a year, when the ground is dry enough for the usage of a tractor but not to dry, so plants are not getting damaged because of the drought. Only then, it is possible to spray against crop diseases of local importance. The conditions to do this are really fairly specific. There should be no rain at the moment and no rain expected for the next 12 to 14 hours at wind speed forces of 1-3.
For crops in greenhouses, there is more flexibility, but the number of sprays is aimed to be minimized because of two reasons. Firstly, the cost of spraying, the staff and the fuel should be minimized. And secondly, crops with fewer treatments of reagents like fungicides are more attractive for supermarkets. Prof. Brown also gave a distinct for this development: During the 34 years of his career the average number of sprays applied per year to blackcurrants, an important crop where he lives, dropped from 37 to one.
Moreover, there has been concerns about negative, non-specific effects of fungicides on soil microbiota but far to less research on the subject. Prof. Brown portrayed, that he would consider a larger effect of the fungicides very unlikely. The amount of fungicides sprayed per unit area is very low. Furthermore, fungicides are usually applied when the crop canopy is well-formed and the great majority of the spray lands on the plant.
While there are some advantageous in having a very specific targeted method to fight certain crop pathogens, there are also define disadvantageous. It is important to know when such a system would be beneficial and when it would not be, Prof. Brown concluded.
The biggest current concern, regarding the off-target effects of fungicides is that triazole fungicides, which are targeting ERG11 (CYP51) in the ergosterol synthesis pathway in fungi, can have off-target effects on at least one enzyme involved in the hormone synthesis in mammals. These effects are still very small though and far below the level detectable by epidemiological analysis. The EU legislation still requires strictly no off-target effects on the mammalian reproductive system, by whatever method of testing.
This behaviour has made the companies, that develop new fungicides, very nervous, since the increasing threat of losing huge amounts of invested money because of unknown off-target effects. Thereby, a system like ours would have to prove that it does not have any off-target biological and biochemical processes, especially regarding the mammalian reproductive system in any way.
Since the interest in fungicide analogues is quite high, a range of methods have been suggested. But these are most often not as effective as the fungicides and for example require multiple applications on the field. Also transporting and application cost would increase.
Further very important characteristics of our method would be its efficacy and therefore the likely cost of the application. Additionally, a reinfection of the plant after the application should be expected. Also, it would be important to know how long our Troygenics would take to degrade and thereby how many applications would be necessary to effectively protect the crop.
First contact 09.09
Phone call 10.09
Video conference 13.09
Paul Zabel works at a Research Associate for the German Aerospace Center (DLR) in the Space Segment Systems Analysis department and has been part of a unique pilot experiment, the EDEN ISS project. The objective of this project, founded by the European Commission under the Horizon 2020 research programme, was to test a special laboratory suited for plant breeding and food production on future space missions. To test this laboratory under extreme conditions Paul Zabel spent one year in Antarctica near the AWI's Neumayer Station III to cultivate vegetables and herbs.
We approached him to receive an evaluation on the impact of fungi under these special conditions, as well as the precautions that have to be taken to prevent plant damaging fungi.
After we called Paul Zabel, we organised a video conference to discuss the role of fungi during his expedition and some aspects of our project.
There have been two shifts on the EDEN ISS since the start of the project, one in 2018 and one in 2019. A season lasts from February to November. Paul Zabel spent the very first season at the South Pole for this project.
In both years, there have been problems with fungi on the station. During the first months of its use the fungi display a faster growth and can form biofilms in the nutrient solutions of the plants. Under these conditions, fungi can start to grow on moist parts of pipes and uneven surfaces. Over time the growth of the fungi reaches a point of self-regulation. During both years, chlorine cleaner was used to counteract this problem with only moderate success. In the next years, the usage of such methods is planned to be reduced to a minimum. Samples from plant surfaces and the water have been taken and are being analyzed by the Astrobiology Group of the Institute of Aerospace Medicine in Cologne. Still the general burden of fungi on vegetables produced in the Antarctic has shown to be 1000 – 10000 times smaller than the one of store bought vegetables. Analyzed contamination samples at the arctic greenhouse rather contained spores of fungi than bacteria.
For the plants, these fungi do not pose severe threats, only the optics of some plants are affected by the fungi. But beyond that, pipes and filters can become clogged through the biofilms and can cause technical issues. Also, the plants are growing in special cubes out of mineral wool that can become overgrown by fungi. This can potentially lead to the stem of the plants to soften up. As a result, the stems of healthy plants can break and the plant dies. Still this issue only affects a small number of plants per year.
At the moment, the seeds are not being sterilized or treated in any special way, since most of the surroundings are clean surfaces, also the cubes of mineral wool are being heat-treated before use.
It is still under discussion, how extensive the sterilization process for later space missions would have to be. Since, on the International Space Station (ISS), everything is mandatory being sterilized and there are still fungal contaminations occurring in space, Zabel reports. Besides that, growing plants can not be sterilized. In the Antarctic, people are visiting the greenhouse with protective clothes, are not allowed to touch anything and have to follow certain rules like “no food” as an “intermediate solution”. This approach could be adapted for space missions later on. Cleaning and sterilization procedures are time-consuming and expensive and will be avoided if possible. In addition to this, vapors of cleaning agents would have to be filtered out in space.
The question arises, if a sterilization of a garden in space would even be possible. For example, on the ISS, four salads have been grown, sterilized with chlorine solution and water and eaten. But it is a rather minor effort to sterilize four salads. The greenhouse Pail Zabel worked in has a base area of 12,5 m2 and would be almost impossible to completely sanitize. Moreover, the water consumption of the sterilization process is quite high. To sterilize 1,5 kg of rocket (salad) around 60-70 l of water are needed, although the salads shelf life expands for about one or two weeks treated this way. So, it has to be evaluated, if the sterilization process would be worth all of this and if the approach sterilizing everything on space missions could actually be continued on this level. After all, the vegetables grown in space could still be eaten without any sterilization process. Maybe the rules on this have to be loosened a little bit, Paul Zabel concludes.
Tests have been conducted with ozone lead into the nutrient solution to lower the growth of fungi. But this has led to the binding and flocculation of nutrients like Iron or Calcium, making them inaccessible for the plants. For future mission in the Antarctic, further measures are already planned. Like tests regarding the sterilization of the seeds and the usage of hydrogen peroxide solution with silver ions added. Also, biological approaches are planned to promote a natural balance and the usage of other strains of fungi to keep the harmful ones at bay could also be a potential measure.
One thing Paul Zabel also pointed out, was that the need for the sterilization of the plants would considerably limit the selection of the plants, since the plant surface has to be accessible for the fungicide. While a salad is quite easy to sterilize, this is almost impossible for crops like carrots or potatoes because they need to be in direct contact with the nutrient solution. Research on soil-free cultivation methods of these plants is being conducted but showed to be rather challenging.
Keeping the current situation of plant breeding in space in mind, there could actually be an application of our Troygenics for future space missions. Since the ultimate goal of a colony on, for example, Mars would be the total self-sufficiency of the colony, the supply on reagents to fight plant pathogens would be extremely limited. A possibility to produce targeted reagents, that only work on the fungi that are harming the plant without harming any of the beneficial fungi for the plant or the plant itself could be a huge benefit. Furthermore, our Troygenics could be modified, adapted and produced in a laboratory in space using E. coli. As a result, the colony could operate independent from supplies from earth. Since the dependency on the own harvest would be way more important in a self-sufficient space colony and the loss of plants through pathogens, fungi, or even radiation in space should be limited to an absolute minimum, our system could contribute to ensure the protection of crops under these conditions. Avoiding technical difficulties, like clogged pipes or filters would be another important issue, since repair parts are often not available.
The ability to manufacture tools or measures to solve problems, like the Troygenics, to assure the self-sufficiency of the colony in space is extremely important, Paul Zabel resumes.
First contact 25.06
Phone call 19.08
We contacted Prof. Dr. Karl-Heinz Kogel, head of the institute for phytopathology at the University of Giessen (Germany), to gather additional information on fungal crop pathogens, their impact and their ecological and economic value.
After we explained what the iGEM competition is and our project we discussed the different parts of our work.
Prof. Kogel pointed out, that our project has to be planned with a lot of foresight, since the current political and social opinion on genetic engineering would prevent such an application in the near future. With the legal situation regarding this topic in the EU he did not expect a system like our Troygenics to be applicable anytime soon.
Besides that, he still liked our approach and considered it a creative idea. Although, he also mentioned, that most comparable approaches do not try to solve this many problems at once, which makes our project susceptible to more problems in direct comparison.
He was especially interested in our approach of an induced endocytic uptake into the pathogenic fungi via specific ligands on the surface of the Troygenics.
As an additional, similar approach he proposed the idea of using only dsRNA instead of the Troygenics and gene silencing instead of CRISPR/ Cas. While this concept would elute some of the problems of our system, like the required uptake of, in comparison, larger particles into the target cell, it would also establish new ones. On one hand this would prevent GMOs to form on the fields. This would make it more realistic to implement and is closer to the current praxis. On the other hand, this would require the use of siRNAs which are distinctly more instable and way larger amounts of them would be needed to achieve the same effect. Moreover, this alternative approach would lack the shuttle our concept provides. Finally, the biological production of the Troygenics via E. coli would be a clear advantage in direct comparison.
Although, this was a great suggestion, we still considered our approach as a better solution to generate a broader range of potential applications.
Furthermore, Prof. Kogel gave us additional information about pathogenic crop fungi and their impact on agriculture. As a further potential target, that would be important to come by, he named Fusarium graminearum as an important pest of wheat and corn plants all around the world. He even mentioned some genes of certain pathways to target for our system. In this case, the pathogen usually is treated with azole-based pesticides, which deactivate proteins essential for the infection of the plant. F. graminearum has become a substantial threat to the food security of the named crops.
Regarding Puccinia graminis, a devastating wheat pathogen, that managed to develop multiresistant strains and is threatening the food supply in parts of Africa and that is spreading continuously, he confirmed the danger of the pathogen and added, that its impact is going to increase dramatically because of the progressing climate change.
Prof. Kogel also confirmed, that problems due to fungicide resistance are already a relevant threat to our food security and supply. A far range of papers are published about this topic.
At the end, we asked Prof. Kogel about methods to distinguish between different spores of fungi and to determine their species without sequencing parts of their genome. Prof. Kogel explained, that the morphology of the spores of different fungal species are quite easy to distinguish with a closer investigation via microscopy.
First contact 04.10
Phone call 16.10
We approached Prof. Haberlah-Korr because of her extensive knowledge about plant protection and ecology and wanted to ask her about the impact fungicides have on ecosystems.
Prof. Haberlah-Korr pointed out, that the impact of the fungicide depends massively on the kind of fungicide that is used as well as its mode of action. Besides that, fungicides can have negative side effects regardless of if they are biological or chemical reagents. Biological alternatives like copper particles are widely used. These particles can accumulate in the soil but are not harmful for humans in the doses they are used in.
A very broad range of fungicides is used with very different modes of action. Some of them can have insecticidal off-target effects or can have negative impacts on soil or water systems.
Despite the public opinion, fungicides do not have a massive negative influence if they are used properly. Besides that, their usage can be justified in many cases, since our level of food production would not be feasible without them.
Many fungicides work quite specific and do not pose a threat through off-target effects. Others, like many azoles, have a broader mode of action and can even show endocrine effects in mammals. Recently, many fungicides have been revaluated because of stricter regulatory limits, but still for example endocrine effects are difficult to measure.
The Federal Office of Consumer Protection and Food Safety monitors the effects of different fungicides and their potential hazards.
Meanwhile, farmers often try to avoid fungicides by more carefully choosing their crop strains or are using resistant plants.
First Contact 05.09
We reached out to Dr. Singh as a contact at the International Maize and Wheat Improvement Center (CIMMYT), an international non-profit agricultural research and training organization that is connecting scientists and research programs worldwide to advance in crop protection for the two most important cereal grains in the world: maize and wheat.
Dr. Singh pointed out, that the International Maize and Wheat Improvement Center has mainly set its focus on keeping damages through fungal pathogens under control by using host resistances of the plants. Furthermore, fungicides are rarely used by smaller farmers in Asia or Africa. Keeping this in mind, the usage of our Troygenics would be quite complicated to realise in those African and Asian regions, since there is no infrastructure to distribute our system in an easy way. Also, our Troygenics are designed in a way, that they could be applied with the tools that are commonly used by farmers to apply fungicides, but without them being used, distributing the respective tools would be an additional problem.
Another point he criticized, was that the targeting and neutralizing of one single fungal pathogen would not avert the threat of fungal damages for the crop. Because of the high variety of fungal pathogens in a country or across countries. To effectively protect the crops a broader applicability would probably be necessary, he concludes. Beyond that, in his opinion this method would have to be implemented into the host plant but like our system, this would be genetic engineering and Dr. Singh did not expect such a measure, based on transgenic organisms, to be applicable in the real world, since the legal situation would clearly prevent such a system from being accessible for farmers.
First contact 13.08
Phone call 15.08
We approached Prof. Dr. Gabi Krczal because as the director of AlPlanta, the Institute of plant research (Neustadt an der Weinstrasse), and former head of department of integrated plant protection in Mainz and former leader of the “Center of green genetic engineering” in Neustadt we really valued her evaluation of our project.
During a phone call, we discussed different parts of our novel approach.
Prof. Krczal considered our system as a sensible approach to reach our goal of transforming pathogenic fungi. Besides that, she also mentioned a lot of things that have to taken into consideration for the successful commercialization of our system.
For example, the price of our system should not dramatically exceed the price of similar, commonly used reagents, unless we would pose some drastic advantages. Moreover, these kinds of reagents would have to undergo tedious testing processes to be used in agriculture. The legal standards in agriculture are high, even higher than the ones applied in the testing of new pharmaceuticals. To receive an official approval for a new reagent of this kind an investment of about ten million Euro would be considered as normal expenses. Of course, the process of approval would also include sophisticated legal assessments and since using genetic engineering is seen rather critically in the EU it would be hard to realize.
Regarding agricultural genetic engineering Prof. Krczal stated that the overall perception of this topic is rather a negative one. But, although the public opinion is mostly against using these methods, genetically altered animal feed is still allowed in Germany. In general, the development of this topic can be described as kind of stagnated in Germany, Prof. Krczal depicts the situation. For example, more than half of the European countries positioned themselves in favor of these new methods while Germany has abstained from the vote. The government of the Netherlands repeatedly tried to permit using genetically engineered products in the EU but was not successful.
Because technologies like CRISPR have such a bad reputation in the EU, countries like Germany could encounter problems if they try to import products that have been altered at any point using these techniques. Because of this, the USA already signalized, that they would get the WTO involved to open up the German market for selling these products.
Regarding our project, Prof. Krczal stated that it would be an important advantage if our Troygenics would be applicable together with commonly used methods for similar reagents. She hinted that some fungi growing into or inside the plants could pose a technical problem to our system, as they can be hard to reach for substances applied to the outside of the plant.
Upon discussing the specifically of our Troygenics in laboratory environments, Prof. Krczal confirmed that they could be used for specifically fight contaminations in cultivations or the detection of pathogenic fungi. To easily validate that the system works, Prof. Krczal also advised us to use reporter genes to assure an easy detection of successful integration of our system into the targeted organism.
Beyond that, Prof. Krczal named us some politicians to reach out to, who are dealing with the regulations of genetically modified organisms on a national level.
First Contact: 19.10
Skype Conference 02.10
We approached Tessa Alexanian as a member of the iGEM Human Practice Committee to talk about the value of Human Practice for iGEM and beyond. Moreover, we received a general evaluation of our Human Practice.
We talked about the possible risks from an accidental environmental release as well as rights around the world with regards to the release of GMOs, which is something we worked on in our team team before. Afterwards, we talked about different approaches to regulating scientific research and to transfer it to the field. This gave us a great overview about important topics of Human Practice and gave us examples on how Human Practices can be used.
Furthermore, we talked about judging in general and the interaction with judges at the Giant Jamboree. Tessa also explained to us, that integrating Human Practices into your project is often a question of how your work as changed by the interactions with experts etc.. Human Practice efforts should fit into your overall story, she concluded.
Project
First contact 25.06
Skype conference 23.07
We approached apl. Prof. Dr. Ulrich Schaffrath from the Department of Plant Physiology at RWTH Aachen University to gather further information about pathogenic fungi and their impact on different aspects of society. Besides that, we hoped for an evaluation of our project and some advice for the optimization of our early concepts.
We discussed our project and some questions during a Skype conference.
The working group of Prof. Schaffrath is doing research on pathogenic fungi that damage crops like cereals, for example wheat, barley and rice. Asian Soybean Rust is of especially high interest because it has the largest economic impact. A part of his research group is also working on the transformation of fungi.
Prof. Schaffrath pointed out, that fungicide resistance has been an underrated problem for quite some time now. Meanwhile, resistances have become an even bigger problem and new strategies to fight them are urgently searched for.
For example, the plant pathogen stripe rust diminishes the area of chlorophyll of the plant and thereby reduces its yield. Because they need lower temperatures, they only spread around the UK but meanwhile a new strain emerged that also thrives under the warmer climate in countries like Germany.
Furthermore, the wheat stem rust strain Ug99 had overcome all resistances implemented into plants that have been used against it and poses a threat to whole harvests in all affected areas.
A new counter strategy for the fight against pathogenic fungi, is the usage of a mixture between different reagents and coordinated usage of different fungicides at different times.
Research has shown, that only a single nucleotide mutation is required to potentially gain resistance in pathogens against a fungicide. Because of this, resistances can emerge after one or two years of fungicide usage.
Because only one base pair has to change to overcome such a pesticide, pathogens like wheat blast cannot be treated effectively with fungicides, which has led to huge problems in Brazil and other parts of the world. These pathogens make the necessity of new approaches even more urgent.
Regarding the realisation of our system, Prof. Schaffrath described the legal situation in Germany and the EU as the biggest barrier.
For the induced uptake via endocytosis he indicated that finding the right surface ligands will be one of the hardest tasks to accomplish, since many pathogens are not well researched. Moreover, many pathogenic fungi are quite hard to study in the lab. The wheat stem rust (Puccinia graminis f.sp tritici) for example is an obligatory biotrophic organism which cannot be grown in a petri dish. After we focussed on P. graminis at the beginning of our project, we shifted our attention on other pathogens after our discussion with Prof. Schaffrath.
While achieving specificity through the surface ligands, can be difficult to achieve for some species, accomplishing specificity through the Cas 13a is more likely to be successful, since many fungal genomes are characterised quite well. We decided to make this part the most important one to assure the specificity of our Troygenics, additional to the other measures like the specific ligands.
Another approach we discussed, was the idea to use mycoviruses as alternatives to our Troygenics. Prof. Schaffrath mentioned to not be an expert in this field, but we still managed to evaluate this topic. Since mycoviruses are not transmitted easily between fungi but rather through the fusion of hyphae and asexual reproduction their uptake would be rather difficult to achieve. If the mycoviruses would be sprayed onto the fields like fungicides or the Troygenics, the uptake would be estimated to be rather low in comparison.
Another topic we talked about was, if the usage of fungicides or the usage of pathogen resistant plants would be more effective to which he replied, that both are important measures of agriculture and are equally useful and necessary. Resistance genes interacting with pathogen proteins by protein-protein-interaction can be overcome by the pathogen quite easily by point mutations. Therefore, plant breeders began stacking resistance genes to lower the risk of these preventive measures losing their effect. Because plant breeding requires a lot of time, fungicides are often needed as a faster counter measure against new pathogens. However, fungicides might impact the environment and therefore must be used carefully and new, more precise versions of fungicides have to be developed. Furthermore, to lower the negative impact on the environment modern pesticides are applied at very low concentrations to fit the changing legal situation. The optimal solution to this would be perfectly working resistant plants, but for now, fungicides are still required. A well-managed mixture of both measures should be the optimal approach nowadays.
We also talked about genetic engineering and its public perception and concluded, that the public discussion is often held on an emotional level. While many scientists are in favour of genetic engineering, it has also become a topic often misleadingly used for politics. The unease about genetic engineering should be evaluated critically, as people often disapprove genetically altered food while openly accepting new medications based on these methods. Moreover, there are products accepted by the society that have been produced using genetically altered organisms, like recombinant proteins, which are used in the production of cheese. However, these proteins do not require to be declared as genetic engineering.
To preserve our abundant food supply, genetic engineering would represent an important tool.
For our proof of concept, we wanted to know the most common strain of wheat in our area to conduct our experiments under realistic conditions. According to Prof. Schaffrath, there is no such thing as the one most common strain of wheat for Germany. Instead, the optimal strain varies locally and from year to year as well as regarding to the location in comparison to, for example its distance to the coast. He also advised us on contacting a plant breeding companies like KWS to get access to wheat seeds, which we implemented later on during our project.
As further important crop pathogens he named Phytophthora infestans, the potato blight, and Asian Soybean Rust but also some bacteria and crop damaging insects. Besides that, he explained that fungi can have a very negative influence on trees as well. There are fungi that can destabilize trees and endanger whole species. Further, trees in cities have to be cut down regularly due to these fungi infections.
Regarding pathogenic fungi for humans, he explained the problem is taking a similar course as antibiotic resistant bacteria. He suspected a higher acceptance of our system as in a medical application opposed to an agricultural tool.
First contact 13.08
Phone call 15.08
We approached Prof. Dr. Gabi Krczal because as the director of AlPlanta, the Institute of plant research (Neustadt an der Weinstrasse), and former head of department of integrated plant protection in Mainz and former leader of the “Center of green genetic engineering” in Neustadt we really valued her evaluation of our project.
During a phone call, we discussed different parts of our novel approach.
Prof. Krczal considered our system as a sensible approach to reach our goal of transforming pathogenic fungi. Besides that, she also mentioned a lot of things that have to taken into consideration for the successful commercialization of our system.
For example, the price of our system should not dramatically exceed the price of similar, commonly used reagents, unless we would pose some drastic advantages. Moreover, these kinds of reagents would have to undergo tedious testing processes to be used in agriculture. The legal standards in agriculture are high, even higher than the ones applied in the testing of new pharmaceuticals. To receive an official approval for a new reagent of this kind an investment of about ten million Euro would be considered as normal expenses. Of course, the process of approval would also include sophisticated legal assessments and since using genetic engineering is seen rather critically in the EU it would be hard to realize.
Regarding agricultural genetic engineering Prof. Krczal stated that the overall perception of this topic is rather a negative one. But, although the public opinion is mostly against using these methods, genetically altered animal feed is still allowed in Germany. In general, the development of this topic can be described as kind of stagnated in Germany, Prof. Krczal depicts the situation. For example, more than half of the European countries positioned themselves in favor of these new methods while Germany has abstained from the vote. The government of the Netherlands repeatedly tried to permit using genetically engineered products in the EU but was not successful.
Because technologies like CRISPR have such a bad reputation in the EU, countries like Germany could encounter problems if they try to import products that have been altered at any point using these techniques. Because of this, the USA already signalized, that they would get the WTO involved to open up the German market for selling these products.
Regarding our project, Prof. Krczal stated that it would be an important advantage if our Troygenics would be applicable together with commonly used methods for similar reagents. She hinted that some fungi growing into or inside the plants could pose a technical problem to our system, as they can be hard to reach for substances applied to the outside of the plant.
Upon discussing the specifically of our Troygenics in laboratory environments, Prof. Krczal confirmed that they could be used for specifically fight contaminations in cultivations or the detection of pathogenic fungi. To easily validate that the system works, Prof. Krczal also advised us to use reporter genes to assure an easy detection of successful integration of our system into the targeted organism.
Beyond that, Prof. Krczal named us some politicians to reach out to, who are dealing with the regulations of genetically modified organisms on a national level.
Phone call: 30.8.19 and 17.09.19
We repeatedly talked to Prof. Holger Deising, positioned at the Martin-Luther-Universität Halle-Wittenberg. He advised us to have a look at another fungus: Colletotrichum graminicola. It is a fungus pathogenic for corn and has previously been used to test RNAi systems.
We were curious, whether he thought our system could work in this fungus and he stated that we could just try it out. Following up on this exciting example we discussed all our subsystems and thought about which one we could test for C. graminicola. Upon learning from Prof. Deising, that endocytic uptake is not an issue for using our system in this fungus, we thought it might be interesting if our CeDIS would work.
Discussing this possibility, Prof. Deising mentioned that we could come and visit him in his lab to transform the fungus with our CeDIS, actually enabling us to test it in a real-world corn pathogen.
During our first talk, we discussed the basics of using our system in this fungus: which genes would we want to target, and which promoters should we use to express it.
Since we wanted to use inducible or repressible promoters to be able to properly estimate the efficiency of our CeDIS, allowing us to distinguish whether the fungus just did not grow or got targeted by it, Prof. Deising suggested using an iron-dependent promotor. He also stated that, for this initial test, the genes would not have to be essential: there were some genes he stated he was sure they would be expressed in the conditions we would grow them in.
While we did talk to him two more times and discussed our design adapted to C. graminicola, we sadly did not get to visit him in his lab due to the limited time within the iGEM-competition.
First contact 19.09
Video conference 29.07
Prof. Dr. Russel Cox leads a research group that focusses on the biosynthesis of natural products by fungi using methods of Synthetic Biology. We discussed our project during a Skype conference and evaluated some ideas we had together.
He assured us the importance of projects like ours, since these are essential to face the growing demand for food in the future and to secure the food supply. Besides that, the danger of fungi gaining resistance to fungicides becomes more and more threatening and the impact of fungi on food production or the health care system can be expected to increase drastically in the future due to climate change.
He also affirmed that fungi are of great importance for the Industry of Biotechnology but still have an enormous unused potential. Although they are used on an industrial scale many fungi are still not well established. For this, a new method to accelerate the transformation and selection steps would be an important tool.
The importance of fungi for the industry is increasing drastically in recent times. They are being used in fermentation processes on an industrial scale in sectors like medicine and food production, for example for penicillin.
Since he and his research group are investigating in metabolites, we discussed the usage of toxic metabolites to fight pathogenic fungi. Prof. Dr. Cox estimated, that 4-5 different complex genes would have to be expressed to efficiently work for fungi. Through our discussion he affirmed our plan to use a Cas System for our system, since it would be easier to apply for fungi.
Regarding our lab application, Prof. Dr. Cox mentioned, that most substances can be produced with fungi, although this often requires a huge amount of work. The problem of these processes rather concerns the slow growth rate of fungi, the difficult transformation and the specific integration. Because of this CRISPR is often regarded as the easier choice. Also, research with CRISPR as transformation method for fungi is being conducted. An important goal for improving the work with fungi would be a faster method for transformation.
For our proof of concept, he advised us on thinking about yeast and filamentous fungi in different ways because filamentous fungi are considerably more complex. Filamentous fungi are also becoming increasingly important for industrial processes.
Prof. Dr. Cox especially underlined the fact that our system has to be really specific. A system that would target fungi in general would be disastrous for the ecosystem.
Phone call 21.06
We had an extensive phone call with Prof. Dr. Mark Varrelmann from the Institute for sugar beet research (IfZ) at the University of Göttingen. As the Research group leader for Phytomedicine he was able to give us a lot of insights into the impact crop damaging fungi have on our agriculture and food production in general. He also gave us an overview about the current situation of research for these kinds of fungi and the latest ideas and methods to fight these pathogens.
We discussed our early concept of our project and asked him to evaluate its functionality. According to him our project is feasible even as a completely novel approach, but we have to take some bottlenecks into consideration. All in all, he estimates our project to be very sophisticated and challenging.
The most important problems have advised us to focus on were the Endocytosis into the fungus, the surface ligands for the uptake of our Troygenics and the adaptability to a wider range of fungi, since fungi are an extremely diverse group of organisms.
The Endocytosis is problematic because the cell walls of fungi are considerably more complex than cell membranes of bacteria and resemble a quite selective barrier between the fungus and its surroundings. Often there is little research conducted on the cell walls of certain species of fungi. Dr. Varrelmann considered the endocytosis uptake of larger particles, like our Troygenics as an especially challenging task to achieve.
Furthermore, he estimated, that the specific surface ligands our system is using to initiate specific uptake into the targeted crop pathogen are limited by the current level of research on this field.
Often the molecular composition as well as the function of some fungal cell wall structures is lesser understood than it would be necessary for our project. So, to realise our project for a larger range of targets, we would have to put some thoughts into how phytopathogenic surface proteins could be identified, since they have not been characterized yet. He highlighted, that a thorough knowledge about surface proteins would be necessary to realise our system as an application for a special fungus. He also added, that genome databases often only contain a small amount of information about phytopathogens.
Most importantly, Dr. Varrelmann advised us to extend our proof of concept with yeast by an additional filamentous fungus to reduce the difference to a, mostly filamentous, pathogenic fungi. Therefore, he suggested Aspergillus niger for this addition, as it represents a well-studied member of the filamentous fungi and has a broad application in the biotechnology industry. Since we are aiming to create a new lab application based on our system to transform fungi and the potential targeting of pathogenic fungi for humans, like some Aspergilli species, a model organism closer to a pathogenic fungus is crucial for out project. Therefore, we decided to integrate Dr. Varrelmanns suggestion into our project.
Last, but not least, Dr. Varrelmann introduced us to several different fungal crop pathogens of economic importance and lead us to further research about this. Moreover, he put us into contact with other molecular mycologists that helped us to optimise our project.
First contact 07.08
Multiple contacts via e-mail and phone for organizational purposes
We reached out to Mrs. Annika Roos who is the marketing consultant for wheats in our part of the country at KWS Lochow GmbH, a leading distributor of seeds for agriculture in Germany and beyond.
For our proof-of-concept, we were planning to cultivate wheat under lab conditions to conduct some tests on the plants. To recreate conditions as close to the regional agriculture as possible, we wanted to find out which strain of wheat is the most common and resilient, so we could use it for our project.
Annika Roos taught us, that there is no such thing as the the most used wheat strain in Germany. Due to differing weather conditions, soil composition or general demands on wheat strains, the choice of the optimal wheat strain has to be taken by every farmer, each year independently and often relies on complex consultancy to optimize said choice.
After this clarification, Mrs. Roos helped us to make a decision on which wheat strains would fit our needs best and which factors and characteristics have to be taken into consideration for this decision. One of these factors would be whether we should use pickeled or un-pickeled seeds.
Ultimately, Mrs. Roos got us access to the strains we decided to use. We received three different strains, each one stained and un-stained. We got the strains “KWS Talent”, “KWS Emerick” and “KWS Fontas” who differ in the quality of the harvest, the yield and their susceptibility to weather or potential fungicides respectively.
First contact 14.08
Skype conference 15.08
We first got in touch with Prof. Richard Oliver through the forwarding of Professor Peter Langridge of the Wheat Initiative. As Professor of Agriculture at the CCDM, the Centre for Crop Disease Management at Curtin University (Australia), Prof. Richard Oliver had accumulated some knowledge we could use to optimize our project.
Regarding the bottleneck of endocytosis that many experts had mentioned before, he pointed out that researchers had thought about using endocytic uptake in fungi before and that there had been a focus in research on this since.
He considered our approach as feasible, since fungi can take up larger molecules from their surroundings. Binding one of these molecules to our Troygenic could thereby, theoretically, induce its endocytic uptake. For comparison, Prof. Richard Oliver mentioned the cholera toxin, which, in a similar manner, consists out of two parts that induce the uptake by the cell and respectively its toxicity.
We investigated the alternative CeDIS idea of introducing siRNAs into our system, but after discussing it we discarded the idea. We did not expect this approach to work in all species of fungi and moreover, this would not allow us to create a lab application, that we are aiming for with our Troygenics.
Regarding other aspects of our CeDIS, Prof. Richard Oliver advised us to increase the number of genetical targets to prevent the targeted fungi from circumventing our Troygenics through mutation. He suggested to use three different targets to assure the applicability of our system. As a result, of this recommendation we also focused our modeling on the question of the optimal amount of gRNAs for our system.
Moreover, Prof. Richard Oliver gave us insights into the combination of methods like fungicides, analogous reagents and plant breeding to protect harvests. He advised us to come up with a similar concept, to assure the integrated functionality of our approach. To proceed with this idea, he also named us a broad range of further experts and associations to get in contact with.
As a potential formulation he suggested using clay nanoparticles that could be attached to our Troygenics and applied to the field. He pointed out that, since they are cost-effective and widely applicable materials, they could be of great use for our project. Contrary to this, we discussed emulating the usage and formulation of currently used fungicides to keep the amount of necessary adaptation to apply our system minimal. However, designing a clay nanoparticle-based system to achieve a slow release system of the Troygenics could open whole new possibilities and potential new applications for our project.
Another addition he made addressed our proof of concept and the verification of the functionality of our system. To investigate the effects our Troygenics would have on plants, we bred wheat plants ourselves to apply our system on them in a controlled environment. In that case we should be able to evaluate the effects of our applied Troygenics based on the visible changes of the plants. For this experiment Prof. Richard Oliver advised us to use specially altered strains of Arabidopsis thaliana with fluorescent reporter genes for the detection of physical damage to the plant. Using these special strains would improve the reliability and the quantification of the potential damage to the plant and would be more precise than an examination of regular wheat plants by eye. He also gave us some lists where we should be able to find said strains and experts to contact to gain access to them.
Prof. Richard Oliver further advised us to watch out for microscopic defense responses of the plant after our tests. If these should occur the composition of the mixture containing our Troygenic probably has to be reconsidered.
A further optimization we designed with Prof. Richard Oliver and implemented into our work was the purification process for the application on the plants. Since our Troygenics are produced in E. coli, just concentrating the Troygenics after discarding cells and debris and using the concentrated supernatant would be an obvious approach. What we did not take into consideration up to that point was that many cultivation media like LB are containing components from bacteria that could cause a PAMP (Pathogen-associated molecular pattern) mediated immune reaction in the plant. Because of this, applying a direct concentration of the supernatant would probably trigger immune reactions of the plant and would thereby become useless for our application. This was one of the reasons why we decided to purify our Troygenics prior to continue working with them. We also thought about that when improving the purification protocol.
First contact 13.08
First contact 20.08
Skype conference 26.08
We came into contact with Prof. Robert Park after a forwarding of one of the other experts of the wheat initiative we discussed our project with. Prof. Park is conducting research about cereal rust and sustainable agriculture.
Regarding our project, we discussed different aspects of our work as well as the problems we had encountered. As we considered the endocytosis as the most important bottleneck of our project, he suggested to use mating proteins to initiate endocytosis. Since we had been using them for yeasts, he stated that these could not only work for those, but that they also exist in rust, even though they do not use sexual recombination. There stil would be further research necessary to determine if this would a valid approach. He also suggested further literature to read about this. Since there is only little published on rust, like he pointed out himself, this has been very helpful.
He also named a set of further potential pathogens for our system to target. For example, the pathogens Ustilago and Tilletia that resemble important pathogens for maize and wheat. These two are also known for the research on their mating but, as pathogens, must be handled according to higher biosafety levels. Despite the fact that they do usually live as parasites, these fungi can be grown in artificial media. Furthermore, he suggested some additional fungi for the testing of our system, that rather resembled the crop pathogens we wanted to fight. Since they were rather difficult to obtain, we have not been able to get access to these strains for our project int this short amount of time.
Prof. Robert Park also gave us general information about his research on rust pathogens and explained, that Mildew or Septoria are having comebacks lately because of their insensitivities to many commonly used reagents.
Together, we discussed the suitability of different fungi for our proof of concept but ultimately decided to stick with Aspergillus for the characterization and later adapt the system to fungi, closer related to the actual crop-damaging fungi. This discussion also settled our choice of using Aspergillus and not adding further fungi with a closer resemblance of actual crop pathogens, mostly due to time constraints.
As an addition, Prof. Park pointed out, that some fungi are dikaryotic and getting the Cas into both nuclei would probably decrease the effectiveness of the system, since our Cas do not has to enter the nucleus, this would not apply to our system.
For our modeling Prof. Robert Park evaluated the parameters we planned to take into consideration and underlined, that the actual rate of mutations in rust fungi is really hard to estimate, since the number of random mutations can vary significantly and depends on a lot of different factors. Moreover, many essential genes would be unsuitable for a targeting due to the rapid evolution of rust fungi, he stated. He also gave us an overview about different approaches of models, that have been developed regarding pathogenic fungi.
For our project, he highlighted the importance of the specificity to assure the biosafety of our application. It is really important to reassure people, that the new application does not damage things unintentionally. The public perception is extremely important for these kinds of approaches.
At the end, Prof. Park underlined once more, that while more research has to be conducted on rust fungi prior, projects like ours are going to have an important impact in the future since the knowledge about these pathogens increases constantly. He pointed out, that our system cold really have a positive impact. We just need some further research.
First contact 22.08
Skype conference 27. 08
Mail with helpful papers attached: 30.8.
In August, we started extensive research on receptor specific endocytosis in Aspergillus niger. Prof. Reinhard Fisher forwarded us to Prof. George Diallinas. At the end of August we had a very informative skype conference. Prof. Diallinas enlightened us about the different Aspergillus-strains and recommended that we should proof our concept in related strains like Aspergillus niduland. He stated, that principles, that work in nidulans will work in niger, too. A disadvantage of niger Diallinas pointed out was, that this strain lacks a sexual cycle so the usage of mating pheromones, that worked properly for S. cerevisiae, was no possible option.
Together with Prof. Diallinas we worked out it would be a promising option to use a virus-like approach. He told us that many viruses use cell-specific transporters to get actively internalized by the target cell. Although he stated that he has little expertise on mycoviruses, he suggested us to target PrnB, an aspergillus-specific proline transporter, with a short proline-peptide fused to our Troygenics.
On the 30th of August, Prof. Diallinas forwarded us some very useful and interesting papers on viruses and how they exploit cellular transporters to get inside their target cell which helped us to design an endocytosis-inducing fusion-protein for A. niger.
First contact 13.08
First contact 25.06
Skype conference 19.08
We established contact to Prof. Dr. Matthias Hahn to receive an evaluation of our project and to gather further information on plant pathogen interaction. Prof. Hahn send us some interesting papers and even parts of his lectures. Later, we also had a Skype conference to ask more complex questions.
As a phytopathologist, we mainly asked him about the uptake of the Troygenics by the pathogenic fungi. In his opinion, the specific, endocytotic uptake resembled the most critical part of the project, because the cell walls of fungi are quite hard to overcome.
While our concept was considered a good idea, we also discussed other approaches. For example, we discussed methods using mycoviruses for transformation through the fusion of hyphen or the interaction between the plant and the fungus through RNAi and exosomes. Though these methods show some benefits they did not featured the same rate of uptake or specificity as our concept.
Overall, he highlighted the need of novel approaches to fight such pathogens and the increasing demand of the industry for them.
We also talked about early ideas for our modelling with Prof. Hahn. In his opinion, predicting the chance of resistances forming is a really difficult thing to do because for natural mutations to occur only one nucleotide has to be altered and every mutation can be dependent on context for itself. Furthermore, we agreed upon the difficulty of testing our hypothesis empirical in the short timeframe of iGEM.
At last, he gave us extended information on metabolic functions of fungi and advised us on implementing a reporter gene into our system, since a validation of our methods via fluorescence would be most convenient to verify.
First contact 02.08
Phone call 06.08
We got forwarded to Fabio Gsaller PhD. by Dr. Alexander Lichius. Both work at the Institute of Molecular Biology at the Medical University of Innsbruck (Austria). Dr. Gsaller has long-standing experience in working with Aspergilli and could advise us on the genetic manipulation of Aspergillus.
During a phone call, we discussed our plans for our project. Although, Dr. Gsaller did not use Cas13a, he had worked with CRISPR/ Cas9 and successfully used the system in Aspergillus fumigatus. While transformation for fungi are quite hard to achieve, it is posible and the protocols for the methods already exist.
He helped us by providing important information on the procedures to transform A. niger. The fungi have to grow for three or four days after the transformation until the colonies become visible. After this, the colonies have to be transferred to new plates multiple times to be sure that they were actually single colonies and overcame the selection marker. Because of this, a transformation process requires two to four weeks, an amount of time that has to be taken into consideration – especially in a competition like iGEM.
As Dr. Gsaller had experience with the transformation of Aspergillus fumigatus and Aspergillus nidulans, he offered to check out our protocols for Aspergillus niger if we would like him to.
He further advised us which lab strain to choose best to and named some scientists we should get into contact with to receive further expertise.
When we discussed our project, he stated that the largest bottleneck within our concept might be endocytosis. Fungi have a cell wall that can be hard to overcome and many therapeutically approaches fail on this task. However, Dr. Gsaller still assumed that the endocytic uptake might work in A. niger if it also worked in closely related strains like A. nidulans.
He thought that our aim should be to create a system where only the gRNAs had to be integrated to adapt the system to a fungus, sincea cas system on its own does not seem to have an impact on Aspergilli.
Moreover, he has sent us further information about A. niger and other fungi that are pathogenic for humans and informed us about their impact. This was important information for evaluating the possible applications of our Troygenics to fight pathogens for humans.
CeDIS
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Lab Application
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Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
