One of the economic pillars of Ecuador is agriculture. Therefore, our team has decided to implement Agrobactory 593 to fight plant diseases using RNA interference technology to silence essential genes of any phytopathogen. One of the most representative export products is bananas. The market is so large that 1 out of every 3 bananas consumed in the world is produced in this country. However, banana production is threatened by the fungus Fusarium oxysporum sp. cubense FOC TR4. Agrobactory 593 will be implemented to combat this disease.
Who are your proposed end-users?
There are about 464 banana-producing companies in the country. In the first instance we target our end users to be medium/small or even large banana producers as a preventive method for the fungus that has caused worldwide devastation. In the future we could expand to other countries where Foc RT4 is also a threat.
Moreover, due to the modularity of our design, there is another group of potential users: Synthetic Biologist. By selecting different target organisms, different genes and different delivery bacteria, the user can customize Agrobactory 593 and use it against many other agricultural pathogens.
How do you envision others using your project?
From a synthetic biologist's point of view, the modular design of our platform will allow Agrobactory 593 to be used for any pathogen of interest. Once the pathogen is selected, the user only has to find the corresponding genes of the pathogen to be silenced. So other users could employ the technology to combat a different pest than the one proposed as proof of concept in our project.
To test the field performance of the dsRNA, the movement of the molecules through the plant will be evaluated. The use of engineered bacteria to produce dsRNA in soil was a key factor in evaluating dispersion through the application zone. Finally, the efficacy of dsRNA will be evaluated by quantifying the number of pathogen conidia and observing the physiological characteristics of the plant, to know if there is any adverse effect on the plant. All this information will be used to train the user on how to use our product.
How would you implement your project in the real world?
Agrobactory 593 is aiming to prevent and treat Fusarium wilt on banana crops. In countries where FOC TR4 is present now, the devastation of these crops has been terrible. Moreover, there is no conventional way to treat this fungus. Chemical pesticides have shown poor efficiency, so farmers prefer to incinerate their crops as a last resort. Additionally, FOC TR4 may become an economic catastrophe since banana crops are one of the main sources of income and employment. Without the use of sophisticated equipment or highly qualified staff the use of dsRNA directly on plantations will prevent the plant disease spreads while reducing logistical problems of distribution.
However, the question remains as to whether dsRNA-producing microorganisms will have any impact on other beneficial plant organisms. It is proposed to solve the above by using a cell lysis mechanism, which controls the bacterial population and avoids competition for nutrients.
In addition, thanks to the technical advice of Antonio Bustamante (see in more detail in Human Practices), we were able to determine in which areas we could deliver our product as a trunk injection.
This scheme represents the zones where the pathogen could propagate. In the center, the Red zone corresponds to the affected plant. The Yellow zone is the buffer or the safety area where the plants are at moderate risk of being attacked by the pathogen.
Our project is targeted to the yellow zone for preventing the fungus propagation towards the Green zone. The green zone is where the pathogen has not propagated yet and the contamination risk is low. Once FOC TR4 is present in the crops, the plant’s quarantine should be extended through all the yellow zone and any production batch. The red zone will be eliminated through incineration to stop the pathogen propagation. Importantly, the yellow zone is an easily accessible area that allows the adequate application of our dsRNA- based biopesticide either by trunk injection or soil spread.
Also, our enterpreneurship team has been working along the lines of a real implementation of the project, you can see here our Sustainable business model.
What are the safety aspects you would need to consider?
One of the key points of Agrobactory593 is the high specificity of the dsRNA produced. Therefore, it does not attack or silence any other organisms that can be beneficial to plants such as Coleoptera or meliponids. Another important aspect to consider is the lysis mechanism of Agrobactory 593. This ensures there will be no remaining live microorganisms that can compete with the soil microbiota for nutrients.
To work with the fungus and avoid accidental contamination or release of Foc-TR4 into the environment, this project has been carried out using Fusarium oxysporum Race 1 (Foc R1), which has homologous genes of Foc TR4. Also, since FOC TR4 is not present in Ecuador yet, we were not be allowed to work with it. The technicians worked in Class II type A1 biosafety cabinet, with the corresponding prior training. They changed their lab coat and protective equipment once they have left the lab.
Bacterial transformation by chemicals, PCR, enzymatic digestion, and chemical RNA extraction were performed under safety protocols. This included working with basic laboratory protocols, the use of gloves for chemical handling and safety, as well as lab coats, to avoid accidents and reagent incidents. E. coli strain HT115 is classified as Risk 1, so the harm caused to humans, animals, and the environment is low.
Read more about this in Safety.
What other challenges would you need to consider?
Each application mode we are considering (detailed in Description) has its own challenges. As for the approach of placing bacteria in the roots that release dsRNA, inadequate spread of the bacteria could trigger off-target effects in the surrounding soils.
FDA approval could be a major challenge because they have to approve the consumption of bananas whose plants have been previously injected or sprayed with dsRNA. In addition, it would be necessary to promote policies to maintain the organic seal of Ecuadorian banana companies. This could be withdrawn when products from genetically modified microorganisms are used.
On the other hand, the application of dsRNA as trunk injection could make it difficult trunk injection could be difficult to perform on individual plants if there are a large number of plants in the crop. Part of the problem was solved by applying dsRNA only within the yellow containment zone, which would reduce the number of plants to be injected.