Team:Groningen/Description
Plant Parasitic Nematodes
Figure 1: Illustration of Plant Parastic Nematodes feeding on the root system. Artist: Kathy Merrifield.
Plant-parasitic nematodes (PPNs) are small parasitic multicellular organisms that live in the soil environment of plants. By feeding on the root system of the plant, these worms take away the nutrients that the plant needs to grow and develop (Figure 1). Together, the nematodes are responsible for a major burden on agriculture worldwide, being responsible for an estimated USD$ 80 billion to 118 billion of crop loss per year (Nicol et al., 2011).
Current solutions include applying inorganic pesticides, last resort methods such as flooding of the field, crop improvement thorugh classical breeding, development of GM crops & crop rotation.
Current Solutions against PPNs
Pesticides
Detrimental off-target effects
Phased out by the EU
Flooding of the Field
Off-target effects
Expensive
Crop rotation
Not fully protective
Not always possible
Classical Breeding
Often not fully protective
Long development time
GM crops
Strict regulations
Reluctant consumers
Inspiration
Figure 2: Potato field affected by Globodera pallida. Plants on the left are treated by conventional methods (fumigation) whereas plants on the right are untreated.
We were first introduced to PPNs by the father of one of our teammates. Kim’s father is a local potato farmer and he explained that small nematodes (which he called “cysteaaltjes") are responsible for crop losses in his fields. After looking into the problem on our own, we discovered that it is the Globodera spp, especially Globodera pallida, that is responsible. Furthermore, we found out that the problem is much bigger than just potatoes. All kinds of crops are currently affected by different nematode species and for most of them, no working and sustainable solution is available.
As a group of mainly Neuroscience and Molecular Biology students, we wanted to create a solution that was in our field of expertise. We developed RootPatch to fight off Globodera pallida from potato plants. However, RootPatch is not only meant to create a solution for Globodera pallida, but a solution that could be applied to different types of nematodes.
Key Values
Before we started planning our project, we set up 4 key values that RootPatch should uphold.
Precision
Safety
Versatility
Easy-to-use
We were aware that there are many other non-parasitic nematodes in the soil that are beneficial to the soil ecosystem. RootPatch should not affect these.
The GMOs of RootPatch should be safely contained at the roots. Therefore, no escape to the environment should be possible.
Not only should RootPatch work for Globodera pallida, but it should also be applicable to other PPNs.
As RootPatch will be a solution that is predominantly used by the farmer, the solution should be easy to use and not too time consuming.
Our focus: Globodera pallida
Figure 3: Distribution of Globodera Pallida. (Source: EPPO global database, www.eppo.int)
Globodera pallida is the PPN with the biggest economic impact on the potato field. In the UK alone, this nematode is responsible for £50 million of losses per year (Leybourne, 2020). Globodera pallida originates from the Andes and over time has spread to 55 countries over the world, most likely due to the distribution of contaminated seed potatoes (Figure 3). In their juvenile form, the nematodes are most detrimental, causing wilting, stunted growth, and other symptoms in the plants that are widely indicative of root damage and stress. The extent of yield loss depends on the population of nematodes, but can in severe cases lead to a complete loss of crops (Crop Science, 2020).
Globodera pallida is a cyst nematode, meaning that its eggs are protected by a strong spherical structure called a cyst (Figure 4). In these cysts, each containing 200-400 eggs, the nematodes are able to survive for up to 20 years. Cysts easily attach to farm equipment but can also spread due to environmental factors such as rain, wind, and floods (CABI, 2019). It is believed that the cysts are the causative agent of the distribution of the Globodera pallida problem. When root exudates of the potato plant enter into the environment of the cysts, the eggs hatch, releasing the nematodes into the soil (1).
Figure 4: a cyst pointed out by Hilde Coolman (HLB)
Figure 5: Schematic representation of the life cycle of Globodera pallida. Numbers in the figure refer to the life cycle steps explained in the text.
The newly emerged second-stage juveniles (2) move through the soil towards the potato roots as they can locate the source of exudate production (Figure 5). After reaching the potato plant, the juveniles start invading the root system (3). Here they use their stylet to manipulate the cells inside the roots, creating so-called syncytia where Globodera pallida drains away the nutrients and water of the plant that it needs to grow and develop (4). After maturation, the female nematodes are fertilized by the males while still in the root system and develop into new cysts containing hundreds of new eggs (5). After a while, these cysts will bud off the roots into the soil where they can stay dormant for up to 20 years and spread to other environments (1).
RootPatch in 4 stages
Application
RootPatch is applied as a powder containing a sporulated bacterium (Bacillus mycoides if proven efficient by our experiments (see Engineering page)). At the start of the season, each potato is inoculated with the powder formulation using farming equipment that farmers are already using. Once the bacteria are in the nutrient-rich environment of the growing potato plant, they will leave their dormant state and start growing a bacterial layer (biofilm) on the developing roots. Because of the high nutritional environment around the plant and the competitive advantage of our bacterium, this biofilm will be maintained throughout the season without any further maintenance. For more information about the application method, check out the Implementation page.
Neuropeptides
To keep the parasitic nematodes away from the potato plant, we will use neuropeptides. Neuropeptides are small neuroactive molecules that play an important role in the neuronal signaling of the nematodes. Modulating the neuropeptide levels inside the nervous system will affect the behavior of the nematodes. We will use neuropeptides from a class called neuropeptide-like proteins (NLPs). These peptides do not require any post-translational modifications and have been shown to affect the nematode's attraction towards the plant root exudates. Several NLPs show the potential to only be effective for specific parasitic nematodes, leaving non-parasitic nematodes in the soil unaffected. Moreover, different NLPs can be used to target different parasitic nematodes.
Click here to learn more about neuropeptide-like proteins in nematodes!
Protecting the plant
To keep Globodera pallida away from the plant, we intend to create an environment with NLPs around the roots. We will do this by having the host bacterium in RootPatch (Bacillus mycoides) produce NLPs. Whenever nematodes come in close proximity of the roots, they will take up the NLPs, which will shift their internal neuropeptide balance. Their chemoattraction towards the root exudate will be affected, causing them to avoid the environment of the plant. This will prevent their infiltration and keep the plant healthy and safe.
Safety
Because we are working with genetically modified microorganisms in the soil, it is very important to consider the safety concerns that come along with such an approach. Setting a genetically modified organism free in the soil is potentially unsafe since damage to the ecosystem is difficult to predict. Bacillus mycoides will be engineered to be dependent on two molecules that are typical for the environment of the potato plant: tryptophan and solanine. Whenever a genetically modified bacterium of RootPatch escapes into the wider environment, it will not be able to survive. In addition, by keeping the bacteria of RootPatch in the potato plant environment, more NLPs will be produced there, which will make RootPatch more effective.
Impact of COVID-19
From wet lab to dry lab
We have been heavily impacted by our university's closure during the COVID-19 pandemic (Figure 6). We soon learned that working on our project in the laboratory was out of the picture, but we persevered nonetheless. From biologists to modelers, we all refocused and endeavored to still show the potential of RootPatch during our quarantine period. None of us had a strong background in biological modeling so we followed courses to learn more about modeling in general and how to set up biological models to answer our questions for RootPatch. In addition, to receive more guidance throughout our iGEM period, we got in touch with Sander van Doorn, an expert in biological modeling. He helped us at several critical steps in the development of the models. Although COVID-19 took away our wet lab plans, we are very satisfied with the new skills that we have developed in biological modeling.
Figure 6: Timeline of our iGEM project with special emphasis on restrictions due to the global spread of COVID-19 .
Our communication as a team drastically changed because of the COVID-19 quarantine. From March until the end of August all of our meetings and social gatherings were online, usually via Zoom. During this period, all university buildings were closed so we generally did all of our iGEM work from home. We kept on track by implementing 3-minute talks by each team member in every weekly meeting. This was an opportunity for each team member to explain what he or she had done the previous week and to update the whole team. We also kept up with team bonding (Figure 7). We used the app House Party to play trivia together as well as created online games just using Microsoft Word. During the period of reduced COVID-19 restrictions in Groningen, we would meet outside for some in-person social activities and, after August, we had access again, but just to an office space, at our university and for only a few team members at a time.
Figure 7: Our team having fun during an online team-activity via Zoom.
