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.