In the past years, the ongoing climate crisis has shown us how the changing environmental conditions put our food production in danger. One of the notable problems in this is the spreading of plant pathogens which can easily ruin the whole crop once it is infected. In the future, the situation will likely get only worse due to globalization enabling the spread and global warming affecting the interaction of crops and pests. (IPPC Secretariat, 2021; Jurozek & von Tiedemann, 2011.)
However, the climate crisis isn’t the only reason to wake up to protect local agriculture at the moment. The recent Covid-19 pandemic reminded everyone about the importance of local production of food and goods. For us living in Finland and in other countries near the current war in Eastern Europe, the question of self-sufficiency has become all the more prominent during the past year. In these conditions, we came to the conclusion that now if ever is the right time to concentrate on helping local agriculture.
This is why we came up with CropFold, a plant pathogen detection system that can be altered to detect several different plant pathogens. Our solution is to help the farmers determine possible infections as soon as possible and to enable early care for the infected crop. This way CropFold is able to help both singular farms to protect their livelihood and the community around them to sustain their resources amidst the changing global situations.
We also believe that CropFold can help reduce the number of pesticides used in the fields. If farmers are able to determine and identify the infection, unnecessary use of pesticides as a precaution can be avoided.
Our CropFold can be used with just a few simple steps (Fig. 1)!
1) Sample collection Samples for CropFold testing can be taken either from plant growth after the planting or seeds before. Samples are crushed before the next steps.
2) NASBA amplification Combine the provided reagents with the sample and heat the solution in a water bath to 65 °C for 5 minutes and then cool it to 41 °C for 5 minutes before adding the enzyme mix provided in the kit. After that, keep incubating the mix at 41 °C for 90 minutes. (Cao et al, 2021; Leone et al, 1997.)
3) Test with the CropFold strip A few drops of the amplification product are added to the test strip.
Wait for the color to change In case of infection, the test strip will change color. The color will show on the test strip in an hour.
Our end product would be distributed as a kit that provides a test strip harboring the toehold switch and a NASBA reaction master mix containing the correct primers for the specific pathogen.
The detection system is based on a cell-free technique mainly to ensure its safe usage. Using cell-free protein expression also helps with the difficulty of getting the viral mRNA from the sample into the detection cells. To enhance the performance of the cell-free system, we decided to add an RNase inhibitor to our kit, as RNases can often cause inhibition in cell-free systems because of RNA degradation (Earl et al, 2017). More about the design can be read on our Design page, and the safety aspects are discussed more on our Safety page and later on this page.
Our test is colorimetric which makes it easy to observe the results without any special equipment as the color change can be seen by the naked eye. This, however, poses a question about the inclusion of possible colorblind users. We answered by assessing different colors with filters demonstrating how the colors are seen by a person with different variations of color blindness. As a result, we found out that the only ones clearly distinguishable with all protanopia, tritanopia, deuteranopia, and monochromacy were the pink created by mScarlet-I and red from beta-galactosidase with CPRG (chlorophenol red-beta-D-galactopyranoside) as a substrate. Therefore, these are the reporters we choose to be used in our test after the initial testing phase. More about inclusivity and the use of colors in our project can be found from our Inclusivity page.
CropFold Kit
- CropFold test strip
- NASBA reagents
- NASBA primers for the pathogen
- RNase inhibitor
- information about the remedies
To improve the storage properties and make distribution easier, the CropFold test reaction will be freeze-dried onto a membrane paper. This enables both longer storage time and storing the test at room temperature. The paper form is also user-friendly as it minimizes the risk of accidentally spilling the reagents and is easier to operate with e.g. gardening gloves than small and flimsy PCR tubes. Unfortunately, we didn’t have time or resources to optimize the NASBA reaction part of the kit, so as of now it would still be done in tubes.
Samples for the test can be collected from both plant growth and seeds (Fig. 2). According to the farmers we discussed with, the growth samples are quite extensive, so one sample per field would likely be enough. For infections that are carried in the seeds the most effective time of screening would be before sowing. The Finnish Food Authority advises to take samples from grains accordingly: take smaller samples regularly from the batch and combine them. Mix well and take a composite sample. (The Finnish Food Authority.) In practice, the samples would be taken by cutting a piece of the plant or the seed and prepared for testing by crushing it well in, for example, a mortar. Other interesting methods would be detecting pathogens from soil or vector insects, such as aphids, but these prospects would still require more research.
As CropFold detects the pathogen mRNA from the samples it’s important to get enough of the genetic material. The mRNA from the collected samples is likely not sufficient to guarantee a reliable result and therefore RNA amplifying should be performed before testing. For the general public without laboratory-grade equipment, we recommend NASBA (Nucleic Acid Sequenced Based Amplification), an isothermal nucleic acid amplification method. Being isothermal, NASBA does not require a thermocycler and consists of fewer steps than e.g. PCR amplification, and is, therefore, easier to implement in field conditions. Nevertheless, the reagents for NASBA are not easily available to people outside of life sciences fields, which is why we decided to include the NASBA reagents in our kit.
NASBA, like PCR, is a primer-dependent nucleic acid amplification method, but unlike in PCR the enzymes used can all work at the same temperature. It is specifically good for RNA amplification as it includes reverse transcription, unlike regular PCR. (Leone et al, 1997.)
To make the usage of CropFold as easy as possible we collaborated with our partnership teams Patras and TecCem to make a detailed handbook to guide the user with the product and other crop quality aspects, such as soil quality.
CropFold started with a clear initiative: to make a detection device for farmers to be used in situ on farms. However, during our Integrated Human Practises work, we realized that CropFold could be applied in other circumstances too.
Part of our Human Practises team visited a local community garden and learned that amateur gardeners might also be interested in the possibility to test their crops.
As plant pathogens such as PVY can spread through seeds (Arce et al, 2021), the most effective time for screening for many crops would be before sowing. This opens up the possibility of testing seeds already before selling them when the testing could be made by the companies selling the seeds. A screening that would include our target viruses is not, at least to our knowledge, done currently for plant seeds here in the EU. Our target viruses are not listed as the 20 priority pests by European Commission (Commission delegated regulation (EU) 1702, 2019), so they are more likely to go under the radar and spread subtly.
CropFold could also help with plant pathogen research and thus research laboratories could also use our system. Their circumstances are significantly different than those of farmers or home gardeners, but our kit could still provide them with a quick and easy alternative to previous methods.
The main application of CropFold is in situ plant pathogen detection for fields and greenhouses. However, the system could also be beneficial for research centers e.g. to get information on the spread of a certain pathogen.
The same cell-free toehold switch technique has been proven to be able to detect human viral infections, such as Zika virus (Pardee et al, 2016) and Respiratory syncytial virus (Cao et al, 2021).
Another beneficial application would be detecting viral infections from insects such as aphids or ticks. This would enable quicker remedies for both plant and human infections where insects act as vectors, such as Lyme disease or potato virus Y.
We decided to make CropFold into a cell-free system to minimize the risks of genetically modified organisms spreading into the environment. Our system does not use any living parts of a cell and the components of our system are not able to spread on their own.
The GMO legislation in Finland, as well as in most of the EU, is strict and all genetically modified organisms have to be authorized on the EU level before they can be distributed commercially or otherwise within the EU (Geenitekniikkalaki, 1995). Thus, a cell-based detection system wouldn’t be viable here under the current legislation. A cell-free system in turn doesn’t contain any components that could spread or propagate and therefore isn’t a GMO referenced in the law. This enables the use of CropFold already under the current legislation.
In all scientific work, the possibility of Dual-Use is always an important thing to consider. The responsibility of careful distribution strategies and overall biosecurity heightens when working with GM organisms, and therefore we are glad to have participated in the iGEM workshop about Biosafety and Biosecurity in Linköping. We want to ensure that our product is promoting health, equity, and a safe environment, and that’s why we need to know where to be extra rigorous.
The most prominent risks are related to distribution and expenses. Misused, our system could contribute to the inequality between farmers of different incomes. If the testing is done before sowing, possibly even before purchasing the seeds, wealthier farmers could afford to buy clean seeds to ensure their future success but the less wealthy would not have the same advantage. Even if testing would be done during the growing season, our test could provide a way to be able to react more quickly to infections, which would cause inequality if expensive.
The next concern relates to malevolent marketing. As CropFold is used to determine infections in crops that are consumed by humans, it could raise false fears about food “cleanliness”. Without a proper understanding of plant infections, consumers might deem all not-tested products as a possible health risk and start expecting unnecessary testing from all of the products sold. This would lead to not only needless panic but also a lot of futile work and a worrying dependency on testing resources.
The last scenarios are those of pure ill will. In theory, our test could be used with the purpose of deliberately transferring the confirmedly infected plant to a new field and causing the spread of the infection. This could be done to specifically ruin someone else's yield or purely to inflict harm in general. Another way of harming others would be to test someone else's crop and state that it’s dangerous in hopes of undermining their sales.
Each research project has its own set of risks with different levels of hazard. In our case, after evaluating the risks we came to the conclusion that the risks in our case are low compared to the potential benefits for the farmers and the communities around them. Therefore we are confident that CropFold would be a product worth actualizing.
We would like to see CropFold reach its full potential in the years to come. We believe that there are still several ways to make our system even better and enable an even larger impact in the future. Due to the limited time frame of the competition, we are not able to conduct all this work on our own but we see this as a good place for a future team to continue.
In the future, we’d be interested to see teams investigate more into testing plant pathogen vectors such as aphids. This would allow the farmers also to diminish the number of pesticides used if they’d be able to make sure the aphids they’re having do not carry a virus.
CropFold is a modular system that allows the testing of several different viruses and could theoretically be used with other pathogens too. We would need new evaluation methods for our different toehold structures to find the most optimal design for our application. The next step after this would be to expand our toehold switch library and conduct experiments to see if bacteria and fungi genomes will work similarly with our system in vitro.
