Team:Groningen/Human Practices
Crop protection/Crop development
We contacted various companies involved in protection of crops against Globodera pallida by either developing products that are applied to the field or selecting new parasite-resistant plant strains. These companies gave us an idea of current approaches against parasitic nematodes and explained the severity of the problem. In general, they revealed to us that there are currently no definite solutions that are sufficient to deal with the problem and that the ones we have are becoming increasingly ineffective because of a changing Globodera pallida population. The companies also explained how they approach the issues and what they believe are the best solutions. More importantly, they gave their honest opinion about our project which provided us with a wealth of information and input that helped shaping RootPatch.
Policy Makers
RootPatch will use a free-living, genetically-modified bacterium in the soil. Although it is meant and designed to be contained at the roots, there are still several safety issues with such an approach. Moreover, RootPatch employs neuropeptide-like proteins, molecules that seem to be very specific, but without proof that that will also be the case in nature. To uncover all possible safety issues in our project, we spoke to members of the National Institute for Public Health and the Environment (RIVM) of the Netherlands. They walked us through all the current guidelines and helped exposing safety risks of RootPatch. This also allowed getting an understanding of what needs to be done before we could actually put a product like RootPatch on the market.
Potato processing
We wanted to get an idea which problems the companies that are selling potato (products) are faced with, especially concerning the GMO discussion. From talking to several companies in the field we learned that potato consumers are reluctant to eat something that has an association with GMOs and that this is especially the case when the product itself is genetically modified. RootPatch may have an advantage over GM crops since it is not the potato but a layer around it that is genetically modified.
Experts
To get guidance on the development of RootPatch, we contacted various (independent) experts working on parasitic nematodes, on crop protection, and genetic on engineering of microorganisms. In addition, because we had to change our entire approach due to the COVID-19 restrictions. We brainstormed with experts in various modeling techniques about what was still possible to investigate in RootPatch. Together, these experts have significantly helped shaping RootPatch as it is now.
Potato farmers
Farmers are central to RootPatch. They are the ones who would have to use RootPatch in the field and possibly, maintain it throughout the season. To learn about all the ins and outs of producing potatoes, their experience with nematodes and to get their opinion about using a GM solution, we visited a number of farms to talk to the owners. These conversations not only showed us their passion for producing the crop, but also gave critical practical insights that helped making RootPatch more user-friendly, especially with respect to how to apply it in the field.
AgBiome
AgBiome is a biotechnology company that is using plant microbiome research for genetic traits and biological pesticides. AgBiome’s products will help farmers combat many of the most important unsolved problems in agriculture, including insects, nematodes, and diseases.
We wanted to know what views they had regarding our project as they have an expertise in the field of plant diseases, and nematodes. Furthermore, since they have a lot of expertise in the type of modeling we wanted to conduct, we wanted their input to help us validate our modeling approach.
We met with Mathew Briggs from AgBiome and discussed our computational model with him. We discussed how we structured our model in terms of differential equations and how we arrived at our values for parameters. Matthew wanted us to articulate clearly what the questions are that our model should answer. He recommended that we keep our model simple and use it to suggest what we will focus our experiments on in the next iGEM year. According to Matthew, this is where our models are the most valuable. He also improved our nematode population model significantly by suggesting an intermediary population of nematodes that are a bit farther away from the roots and are still affected by the neuropeptide-like proteins. He also suggested not to waste too much time on finding exact quantitative values for our parameters but to adjust the model by comparing the end result to existing experimental data. Overall, AgBiome has significantly helped us to improve our modeling approach.
Integrated Human Practices
▪ Bacteria population model: keep it simple, don't make your model too complicated!
▪ Nematode population model: Add an extra nematode population that is "far" away from the plant but is still affected by the neuropeptide-like proteins.
Syngenta
Syngenta is a biotechnology company with the goal to develop more sustainable ways of doing agriculture. They produce agrochemicals, seed technologies, and develop crops that help us produce our crops with a smaller impact on the environment. Currently, they are one of the leading companies in crop protection worldwide.
We met with Martin van Gheluwe and Maxime Lieping from Syngenta and spoke about the different aspects of our project. They were really interested in the use of neuropeptides to protect plants but also pointed out some uncertainties that we have right now. How are the NLPs going to behave in the soil and what will their impact on other organisms be? Following their questions we looked into this and set up protocols that can help us give answers to these questions. In addition, they showed us the business side of creating a product like RootPatch. At the moment, it is impossible to get a similar product on the market. A lot of our current policies in the EU regarding GMOs have to be changed before RootPatch can be introduced to the market.
ECOstyle
We had a meeting with Pier Oosterkamp, head of the company Ecostyle. Ecostyle produces ecological products for the care of all kinds of plants and animals. Among other things, they also deal with soil treatment and sell nematodes to treat certain pests. This is why we wanted to hear Pier’s opinion on our RootPatch approach to possibly refine our plans.
In general, Pier approved of our project and called it a “Modern approach, [which faces], however, legal and economical problems”. According to Pier, the legislation associated with our product would be the biggest problem. You need to register the organism you want to use (in our case, the genetically modified bacteria), but the EU states clearly that it is currently not allowed to release GMOs in the environment. Pier was also concerned about the practical aspects of our product. He was wondering how we would sell our product to farmers and how we would make sure that they know how to apply it correctly. For farmers, GM plants would probably be more efficient, according to Pier.
What we took home from this meeting is that while we cannot do much against the current EU legislation on GMOs, we can make sure that in our product design, the form of application of RootPatch is stated clearly and easily for farmers. In addition, Pier suggested the use of protozoa to improve the chance of survival of bacteria in the initial period after RootPatch application. We implemented this approach in our experimental design.
Integrated Human Practices
▪ Experimental design: Use protozoa to give the bacteria in RootPatch an competitional advantage.
Agrico
Agrico is a potato company involved in various activities related to this important crop. They grow, process, store, retail, and improve potato quality. We spoke with the head of their research department Sjefke Allefs. As Sjefke says: “Cyst nematodes are the biggest problem for Agrico”. No wonder Agrico invests so much into the genetic breeding of the different potato cultivars to develop a new breeds with enhanced resistance towards G. pallida nematodes.
We approached Agrico knowing Sjefke’s long experience in this industry as well as his unprecedented knowledge of the current G. pallida situation in the Netherlands and the common hurdles and regulations in the potato and agricultural market in general. After a presentation at Agrico research facility, we started a discussion about the various aspects of our project and Sjefke showed us around their research facility where they are trying to develop new ways to protect the potato plant.
The discussion with Sjefke provided a lot of insight into the limitations of our current (Aug 12th) project design and the potential challenges on the road to the market. As such, Sjefke considered our models to “have nothing to do with reality” because the soil environment and biodiversity vary so much in different regions that there is no way to make an accurate prediction with our current approach. Sjefke noted that, despite the combination of GM bacteria in soil and neuropeptide production being a “no go” in the current GMO climate, developing a bacterial bioformulation is easier for dossier profiling as it can be applied to any potato variety out there. There are thousands of different potato cultivars but we only need to get RootPatch approved once to protect them all. If RootPatch was developed as a GM plant, it would take too much time and money to protect all the different cultivars.
Sjefke helped us to get an understanding of the current situation and gave us insight into how they are trying to protect the potato crop using classical breeding methods. He made us think more critically about the models we were developing and helped us to set up the key experiments that can help us prove our principle. He also suggested looking at an inoculant product that is already on the market which uses a bacterium species that is capable of surviving at the plant roots. This gave the inspiration to our fourth potential host: Bacillus amyloliquefaciens.
Integrated Human Practices
▪ Experimental design: Explore Bacillus amyloliquefaciens as a potential host for RootPatch.
MicroPEP
MicroPEP is a French crop protection company specializing in natural solutions rather than the chemical ones. They are developing formulations to sustain crop growth, but also to protect the crop against various diseases, and weeds. We reached out to Michael Corbout, CEO of MicroPEP, because of their innovative mentality and we both agree that the use of chemical pesticides needs to be reduced.
In our meeting, we spoke about the EU and US regulations on the use of genetically engineered microbes as bio-based crop protection solutions. Intellectual property concerning NLPs was also an important topic of our discussion, as it appeared to be an important factor for our future potential sponsors to consider. But the take-home message was the importance of a safety mechanism to prevent the spread of B. mycoides.
Integrated Human Practices
▪ Experimental design: Create reliable safety mechanisms that allow the control of RootPatch. Only then would RootPatch be acceptable to the market.
Hudson River Biotechnology
Hudson River Biotechnology is a biotechnology company that specializes in the genetic modification of various crops and microorganisms. They apply their CRISPR technologies to develop novel crop varieties with innovative traits aimed, among others, to protect plants from pests. We spoke with Lotte Westerhof and Ferdinand Los of Hudson River Biotechnology about our project, where they raised various concerns about our approach.
Safety was a major concern for them because we were genetically modifying a microorganism that is free-living in the soil. They mentioned that we need to think about possible horizontal gene transfer between bacteria since our target host bacteria have many similar bacteria in their soil environment. In addition, they raised the idea of running a BLAST sequence with our neuropeptide-like protein to get an indication of possible off-target effects. We implemented the results of this in our implementation page . They also proposed that we should look into the diffusion of the NLPs in the soil, to make sure that we can actually create the NLP environment we desire. Subsequently, we also designed the experiments for this, which you can see on our engineering page .
Integrated Human Practices
▪ Experimental design: Run a BLAST sequence to potentially identify off-target effects of the NLPs.
▪ Experimental design: Look into the behavior of free NLPs in the soil. How fast are they broken down? How far do they diffuse away?
HLB
HLB is a research lab, which has been working in the areas of plant and soil health in agriculture. They specialize in research and recognition of all diseases, plagues, and weeds that affect arable crops, flower bulbs, field vegetable crops, tree nurseries, and sports fields. They also specialize in the research on quarantine organisms.
We decided to contact HLB as our stakeholder because they have been in the plant and crop protecting sector for a really long time. Furthermore, they have expertise in working with Globodera pallida, which can only be studied under special and strict conditions. After an online call, HLB invited us over to take a look at the problem up close in their research facility.
One of the ideas of RootPatch was to implement an extra element that lets the nematode cysts hatch while RootPatch is active. This way we would also get rid of the remaining cysts in the soil, minimalizing the opportunity for the nematodes to spread to other regions. We created a plan that uses the compound DAPG that is known to initiate the hatching of the cysts. However, after conversations with several people of HLB, it became clear that this wouldn’t be necessary since the potato root exudate is much better at doing this than any other compound/mixture. They also helped us in determining our host bacterium, Bacillus mycoides , through one of their regular contacts, Jan Spoelder.
Integrated Human Practices
▪ Experimental design: Don't try to initiate cyst hatching. Root exudate is strong enough on its own.
RIVM
The National Institute for Public Health and the Environment (RIVM) is a governmental department that has a central role to play in disease control. They conduct independent scientific research in the fields of Public Health, Care, Environment, and Safety and hence can be called a trusted advisor to society. We wanted to design RootPatch taking all the safety and precautions in mind and hence, we decided to contact them. We spoke to Cécile van der Vlugt, a senior risk assessor at RIVM.
The feedback that we got from the Cécile was that the regulation needs to be assessed and regarded as safe whenever a new GMO enters the market, but also that the public is not a fan of consuming GMO products as they are still skeptical about it. She explained that food and feed are the major regulations and topic of discussion when it comes to GMO. When we mentioned that RootPatch will coat the potato roots and not genetically modify the plant, she did point out two problems: 1. We should look for food grade, naturally occurring bacteria like B. subtilis and 2. The bacterium is still present on the potato even after harvest.
Lamb Weston
We met with Sjaak Aben from Lamb Weston who is a potato production expert at Lamb Weston, which is a large international potato retailer. Lamb Weston is, therefore, more in touch with the demands of the consumer than the companies that develop new cultivars. Because we had not yet received input from someone who represented the consumers of potatoes, we approached Lamb Weston to receive feedback and learn about the consumer's perspective.
We learned that the consumers are very hesitant of products that are called GMO. However, RootPatch would not make the potato a GMO so that would be a big advantage over the alternative of making the plant itself secrete NLPs. Small differences like these can make a big impact on how consumers view our product. We also learnt that potato cyst nematodes are not the biggest problem in potato production and that resistance to abiotic factors would be more important for increasing yields. The overall feedback about our project was positive in this meeting which made us more confident in the approach we chose for RootPatch.
prof. dr. Oscar Kuipers
Oscar Kuipers is a professor and head of the Department of Molecular Genetics at the University of Groningen. Oscar has been an advisor to previous iGEM teams and has a profound knowledge of microbial systems. We reached out to him to get his input and advice on both the experimental design and computational approaches.
Oscar pointed out that possibly the biggest bottleneck in our project is the survival of the bacteria in the soil. If it gets outcompeted, then the practical applications would be very limited. Furthermore, he suggested we should contact Jan Spoelder about a bacterial strain that could be very suitable for our project. In the end, this became our first choice as a host bacterium for RootPatch. In addition, he suggested talking to Jordi van Gestel, a theoretical evolutionary biologist who has a lot of experience in competition between bacteria. Jordi van Gestel ended up giving us critical information for our computational models.
Integrated Human Practices
▪ Experimental design: Choose a host bacterium with a competitional advantage in the soil.
▪ Modeling approach: Try to investigate competition between bacteria.
Dr. Jan Spoelder
Jan Spoelder is a specialist in classical diagnostics, phytopathology, and in the application of biological agents. He has been working in this field for decades and is currently working in collaboration with the HLB lab.
We contacted Jan after our conversation with Oscar Kuipers. We discussed about a paper which he had supervised which mentions using B. mycoides strain to protect the roots. We were planning to select a chassis and wanted to know if this specific bacterial strain would be ideal for us to use.
Jan suggested a different bacterial strain called M2E15 of B. mycoides and said that we should use this for our project as it is very robust and is a potent colonizer of the potato roots. It is also a general colonizer so it can produce biofilms on any root system. Furthermore, he told us that the GFP variants of M2E15 are available which would help us visualize them. He also helped us in planning protocols for the bioformulation of RootPatch by providing us information on which type of formulation would be ideal.
Integrated Human Practices
▪ Experimental design: provided us with a suitable host organism strain: Bacillus mycoides M2E15
▪ Experimental design: provided the method of application to the potato tubers.
Dr. Jordi van Gestel
Jordi is a Dutch post doctoral researcher in the fields of bacteriology and evolutionary biology. He is a renowned expert in biofilm modeling, bacterial cell differentiation and multicellular organization. We approached Jordi after Prof. Oscar Kuipers recommended us to get in touch with him.
Jordi really helped shape the logic behind our biofilm model. Prior to the meeting, we were in the mindset of creating a complex model of the biofilm at a micro-level (modeling individual cells). Jordi emphasized that when there are several unknown variables in a system, like in our case, a simpler model will provide more robust results than a complex model. He helped us simplify our approach and focus on a macro level biofilm model to be comparable to the nematode population model. He suggested that we describe the average behavior of our bacterium and apply those to the whole population. We can start with humidity and slowly grow in complexity with the competition, and continue with other environmental factors.
With his advice, we were able to set up a set of new equations for our bacteria population model that made more sense and allowed us to describe competition between bacteria. Thanks to the help of Jordi, our model was significantly improved.
Integrated Human Practices
▪ Modeling design: create population based models instead of individual based models to avoid unnecessary complexity.
▪ Modeling design: investigate the effect of environmental conditions.
▪ Modeling design: use the models to guide your attention when performing experiments.
Dr. Sander van Doorn
Sander van Doorn is a professor in theoretical biology at the University of Groningen. Because the COVID-19 restrictions prevented us from going to the lab, we had to change our whole project approach and had to look into alternative options to explore RootPatch.
Sander had the biggest impact on our models of all the other experts we spoke to. He shaped our model from the beginning and explained what was in our reach to investigate with the data from literature. He was the one who suggested doing qualitative analysis on the behaviour of our model instead of trying to estimate quantitative values. After his conversation, we reached out to other experts who helped us shape our model even more.
Integrated Human Practices
▪ Modeling design: perform qualitative analysis instead of quantitative analysis. (parameter sensitivity analysis)
Dr. Harry Teicher
Harry Teicher is an expert in the field of crop protection with 20+ years of experience. He provides professional expertise to innovative agricultural startups on the development and market implementation of their future products. Harry has helped many companies with their product strategy and helped to enter the market.
We contacted Harry hoping that he could give us his expert opinion on the feasibility of RootPatch on the current market, given his long experience. According to Harry, the most innovative part of our project is the safety mechanism that we designed. He said that it is a good feature to consider when developing microbe-based products like ours. However, he was not a big fan of our current approach as he saw the little potential it has of reaching the market. Therefore, he proposed an alternative design (encapsulation of our NLPs) that would be better suited for the current market. This design is added as an alternative strategy to our engineering page.
Integrated Human Practices
▪ Experimental design: consider the option of encapsulation of the NLPs after producing them. in vitro
Anton Varekamp
Anton Varekamp is a farmer in the south of the Netherlands who owns 250 hectares of land which he devotes to potatoes from time to time. We met with Anton at the beginning of our iGEM project when the plan of RootPatch had just taken shape. At this stage, we only had theoretical knowledge about how the plant grows and is maintained. Anton showed us around his farm and explained everything there is to know about growing a potato plant and keeping it healthy and safe throughout the season.
Besides talking about the whole process of potato production, from planting the seed potato to harvesting, we spoke about the regulations that are present in the Netherlands to protect farmers from plant pests such as parasitic nematodes. Every year, before planting his crops, Anton invites a national institute to check his soil for possible pathogens and the overall health of the soil. When he grows seed potatoes, he needs his soil to be checked for potato cyst nematodes. If they find one nematode, he is not allowed to grow seed potatoes until he kills all nematodes. A costly and detrimental procedure.
Anton does not have any experience with the potato cyst nematodes in his field because he is careful. He only grows the potato plant once in 4 years (a method called crop-rotation) so the chance of survival of the potato cyst nematode population in his soil is reduced. However, if it were possible, he would still like to grow the potato plant more often because of the high market demand. His neighbors are doing this, and they are experiencing the consequences of the potato cyst nematodes from time to time.
We spoke with Anton about our project and he told us that he would be interested in using such a product even though it is a GMO. He did point out that the application technique is key. He, and a lot of other farmers, are using machinery that makes it easy to apply a granulate to the field. If we could provide RootPatch as a granulate as well, this would make it much easier for them. Together with the input we got from Jan Spoelder, we indeed decided that RootPatch should be applied as a granulate to the field.
Integrated Human Practices
▪ Experimental design: granulate application of RootPatch would be ideal. Equipment necessary already in place.
▪ Experimental design: RootPatch should maintain itself throughout the season. Multiple applications per season is not ideal.
Pieter van Maldegem
Pieter van Maldegem is a farmer in the province of Groningen, located in the north of the Netherlands. He owns around 75 hectares of land of which 25 hectares are devoted yearly to seed potatoes. These seed potatoes have to be of high quality because they will be replanted. We met with Pieter during the potato planting season. After Pieter showed us around on his farm and in the field, we discussed how RootPatch would be the easiest for farmers to apply during potato planting.
The type of soil in the North of Groningen, in combination with a crop-rotation cycle of 3 years results in a high risk of potato cyst nematode infection. To prevent those infections from happening in his potato fields, Pieter consults the research lab HLB every year to check the soil for cysts. From time to time he also uses potato cyst nematode-resistant varieties to decrease the cyst population in the soil.
Pieter would be very interested in a product like RootPatch. The current methods to decrease the potato cyst nematode population in the field are not effective enough, in his view. The financial loss as a result of a potato cyst nematode infected field is high. If our product could be a solution to the potato cyst nematode problem and was allowed, he would happily buy and apply it. He is not against, or afraid of the use of GMOs in agriculture.
During the visit, we took a close look at the potato planting machine. The potato planting machine used by Pieter contains two tanks, one tank with liquid and a tank for granulate. Both tanks contain solutions to enhance the growth of the seed potato and protect it from various diseases. Just before the potato is planted, liquid and granulate are sprayed on the potato. In addition, the machine used to prepare the ridges in which the potatoes are planted also has a tank. At this ridge preparation step, Pieter adds nutrients to the soil to keep the potato as healthy as possible. We discussed how we could optimize the RootPatch application procedure, and concluded that application in solid form would be more efficient than liquid. In addition, it would be more convenient to apply RootPatch granulate directly on the potato during the planting step than in the soil at the ridge preparation step.
Integrated Human Practices
▪ Experimental design: Solid formulation is efficient to use for farmers.
