Project
Human Practices
Dry Lab
Wet Lab
Jamboree
THAELIA, a bacterial system engineered to fight V. dahliae, has the potential to provide a practical solution to a widespread agricultural issue—Verticillium Wilt. To successfully translate our project into real-world use, we focused on designing an implementation approach that addresses both agricultural needs and environmental safety. Through consultations with agricultural experts, plant biotechnologists, and farmers, we developed an approach that balances effective fungal control with responsible deployment of genetically modified organisms. Our aim is to introduce THAELIA as a biofungicide that benefits crop health and sustainability without harming the surrounding ecosystem.
Through our conversations with stakeholders, the need for a product to cover this particular problem was evident. Our products will essentially be agricultural products, available for farmers to be applied in the open field. This procedure will be facilitated by agronomists, as they will be in charge of the product distribution. We believe that local agronomists, familiar with the farmers’ plight, will be able to pinpoint which product of the two will benefit the selected grove, taking in consideration the local climate and the current conditions, covering practically the entirety of the stressors that would affect product efficiency.
• Defining the problem
Our project, THAELIA, focuses on developing a bacterial system that utilizes bacterial-mediated RNA interference (bmRNAi) to combat Verticillium dahliae, the most dangerous fungi affecting olive trees that causes Verticillium wilt [1]. In regions like Andalusia, Spain, approximately 38-39% of olive crops are affected by this disease, leading to considerable losses in both quantity and quality of olives produced [2]. Given that Andalusia is the world's leading producer of olives, generating around 900,000 tons of olive oil and 380,000 tons of table olives annually from 1.5 million hectares, the economic ramifications of yield losses are important [2]. Even a small percentage decrease in yield can translate into millions of euros in lost revenue for farmers. In Greece, where the climate closely resembles that of Spain's olive-growing regions, V. dahliae has emerged as a significant threat, causing Verticillium Wilt in olive trees, especially in Thessaly. Additionally, the persistence of V. dahliae in the soil limits farmers' ability to rotate crops effectively. Rotate the crops effectively" refers to the practice of systematically changing the types of crops grown in a specific field over different seasons or years in a way that maximizes the benefits of crop rotation. Effective crop rotation helps improve soil health, reduce the risk of pest and disease buildup, and enhance nutrient management [2]. At the same time, the disease affects not only agricultural production but also the nursery trade and landscaping industries that rely on healthy olive trees. Infected nursery stock can lead to disease spread when planted, posing significant risks to both local and international markets [1].
Farmers use a range of common methods to combat Verticillium which often prove ineffective due to limitations related to the biology of the pathogen and the application of current practices. Firstly, chemical fungicides are frequently employed but struggle to control V. dahliae because the fungus resides within the plant's vascular system, making it difficult for fungicides to penetrate and combat the pathogen. Additionally, over-reliance on these chemicals can lead to resistance development, further diminishing their efficacy [3]. At the same time, crop rotation often fails because V. dahliae can survive in the soil for years, waiting for a susceptible host [4]. Moreover, resistant crop varieties may not be available for all plants, and even they can be overwhelmed by high pathogen levels. Farmers who depend on a diverse cropping system for economic stability may also find crop rotation impractical.
In addition, oil testing and quantification methods, like wet and dry plating, are commonly used to assess pathogen levels but often lack precision [4]. These methods can overestimate pathogen presence due to interference from other species in the soil, leading to misguided management decisions. Overall, the ineffectiveness of these methods stems from inaccurate detection techniques, the development of chemical resistance, and the limited success of cultivation practices. As a result, these shortcomings impose significant economic burdens on farmers, especially in regions reliant on olive production.
•A synthetic biology approach
>The result of our efforts is THAELIA, a modular and adaptable bacterial system that combats the fungus using RNAi technology. As outlined in our Project Description, THAELIA produces double-stranded RNA (dsRNA) molecules that specifically target and silence essential genes in V. dahliae, offering an innovative approach to managing the disease. If you want to learn more, you can visit our Project Design page.
•Developing the product
To effectively implement bmRNAi against V. dahliae, we propose two primary methods for the farmers to use: 1) powdered bacteria in an irrigation system and 2) ready-to-use liquid solutions. Each method has distinct advantages and disadvantages that must be carefully considered for successful implementation.
1) Powdered/ Lyophilized Bacteria in Irrigation Systems:
This method involves creating a powdered formulation of our engineering bacteria that can be mixed with water and delivered through existing irrigation systems. The powdered form allows for easy storage, transportation, and potential customization based on olive crops’ requirements [5].
2) Ready-to-Use Solutions:
Ready-to-use solutions are pre-formulated liquid products containing viable bacteria designed for immediate application. This method simplifies the application process, as farmers do not need to prepare the solution themselves, thus saving time and reducing the risk of handling errors [6].
Searching for the most suitable method of application
Method 1: Powdered Bacteria in Irrigation Systems
1) Preparation:
• Develop a powdered formulation of the engineered bacteria capable of producing dsRNA targeting V. dahliae.
• Ensure that the powder maintains high viability during storage and transport.
2) Rehydration Process:
• Mix the powdered bacteria with a nutrient-rich solution before application to enhance recovery and viability.
• Use proper mixing techniques to prevent damage to bacterial cells.
3) Application:
• Incorporate the rehydrated bacterial solution into existing irrigation systems (e.g., drip or sprinkler systems) for uniform distribution.
Method 2: Ready-to-Use Solutions
1) Preparation:
• Formulate a ready-to-use liquid solution containing viable engineered bacteria designed for immediate application.
• Ensure optimal concentrations of dsRNA-producing bacteria for effective pest control.
2) Application:
• Apply directly through irrigation systems or as foliar sprays without additional preparation.
• Provide clear instructions for application timing and methods.
A. Method 1: Powdered Bacteria in an Irrigation System
Lyophilization, or freeze-drying, is a preservation technique used to enhance the viability of bacterial cultures by removing water content while maintaining cell integrity.
Advantages:
1) Cost-Effectiveness:
• Generally less expensive to produce and transport than liquid solutions.
• Longer shelf life reduces waste and costs associated with spoilage.
2) Storage and Handling:
• Easier to store and handle; less risk of contamination compared to liquid forms.
• Lightweight and space-efficient for transportation.
3) Customizability:
• Farmers can adjust the concentration based on specific needs or crop requirements.
• Can be mixed with other amendments or fertilizers for tailored applications.
Disadvantages:
1) Rehydration Complexity:
• Requires proper rehydration techniques to ensure bacterial viability; improper mixing can lead to cell damage.
• Farmers need training on how to properly rehydrate and apply the powder.
2) Application Challenges:
• May require additional equipment (e.g., mixing tanks) for effective application through irrigation systems.
• Risk of clogging in irrigation systems if not properly prepared.
B. Method 2: Ready-to-Use Solutions
Advantages
1) Convenience:
• Ready-to-use solutions eliminate the need for preparation, making them easier for farmers to apply.
• Immediate application without the need for additional mixing or rehydration steps.
2) Consistent Quality:
• Formulated to ensure optimal concentrations of viable bacteria, providing reliable performance.
• Reduced risk of human error during preparation and application.
3) Time-Saving:
• Less time required for preparation allows farmers to focus on other tasks.
• Quick application can be beneficial during critical growth stages.
Disadvantages
1) Higher Cost:
• Typically more expensive than powdered forms due to production and packaging costs.
• A shorter shelf life may lead to increased waste if not used promptly.
2) Limited Customization:
• Less flexibility in adjusting concentrations or combining with other products compared to powdered forms.
3) Storage Requirements:
• Often require refrigeration or special storage conditions to maintain viability, increasing logistical complexity.
| Criteria | Powdered Bacteria | Ready-to-Use Solutions |
|---|---|---|
| Cost | Generally lower | Generally higher |
| Time | Requires preparation time | Immediate use |
| Ease of Implementation | Requires training on rehydration | User-friendly, no additional training needed |
| Storage | Longer shelf life, less sensitive | Shorter shelf life, may require refrigeration |
| Customization | Highly customizable | Limited customization |
| Application | May require additional equipment | Direct application |
Once more, we are faced with a dilemma. However, the best product is not a black and white issue. All problems arisen by the stages and the disadvantages of each process, could potentially translate to an advantage in a unique scenario. After all, no olive grove possesses the exact same conditions that need to be met in order to maximize product efficiency.
This is where agronomists take part. They are the members of the community that know agriculture deep enough to understand the needs of each tree in a different environment. They also share a familiarity with the local farmers, as they are the people where the problem is first mentioned in an attempt to treat it.
Generally speaking, lyophilized bacteria would be a better candidate for hard to reach areas, due to transportation limitations. On the other side, the viable bacteria formulation would be the best choice, as the bacteria would not have gone through the process of rehydration after lyophilization, which is an added stress. In both cases, the development of an additive, formulated for the olive grove’s soil conditions would also be a great way to further establish our bacterial population against local bacterial soil flora. This is another interesting aspect that needs further research!
To prepare the lyophilized engineering bacteria with auxotrophies for tyrosine, Vitamin B12, and Ave1 protein, which are vital for cell proliferation, the following process is conducted:
Cultivation: The bacterial culture is inoculated in a growth medium containing tyrosine, Vitamin B12, and a source of Ave1 protein. The culture is incubated under optimal conditions (appropriate temperature, shaking, etc.), ensuring that the medium supports the auxotrophic bacteria. The growth is monitored, and the culture is allowed to reach the desired growth phase, either exponential or stationary, depending on the objective, such as maximizing biomass or OMV production.
Cell Harvesting: Once the bacteria have reached the target growth phase, the cells are harvested by centrifugation at 4°C to minimize stress and preserve viability. Centrifugation is performed at moderate speeds (4,000-6,000 x g) for 10-15 minutes, producing a bacterial pellet. The supernatant is removed carefully, and the pellet is handled gently to prevent cell damage.
Washing: The bacterial pellet is resuspended in sterile, isotonic phosphate-buffered saline (PBS) or another nutrient-free buffer. The washing step is repeated two to three times, centrifuging at the same conditions between each wash to thoroughly remove residual growth media, ensuring that no tyrosine, Vitamin B12, or Ave1 remains in the bacterial suspension.
Cryoprotectant Addition: The washed bacterial pellet is resuspended in a sterile cryoprotectant solution, such as 10% sucrose or trehalose, to protect the cells during freezing and drying. The solution is mixed gently with the cells to achieve uniform suspension. Cryoprotectants help preserve cell membranes and structures during the freezing process.
Freezing: The bacterial suspension is rapidly frozen, either by flash-freezing in liquid nitrogen or by placing it at -80°C. Quick freezing prevents the formation of large ice crystals, which could damage the bacterial cell membranes. The frozen suspension is then prepared for the lyophilization process.
Lyophilization (Freeze-drying): The frozen bacterial suspension is transferred to a lyophilizer, which is set to a low temperature (typically -50°C to -80°C) and high vacuum. The lyophilizer removes water from the cells through sublimation without allowing ice to melt, preserving cell integrity. The lyophilization process can take several hours to days, depending on the volume and moisture content.
Storage: After lyophilization, the dried bacterial powder is immediately transferred into sterile, airtight containers to prevent exposure to moisture. The containers are stored at 4°C or lower, ensuring the longevity and viability of the lyophilized bacteria. Proper labeling of the containers, including strain information and storage date, is essential for future use.
Rehydration and Use: When needed, the lyophilized bacteria are rehydrated in sterile water or nutrient medium containing tyrosine, Vitamin B12, and Ave1 protein. The bacteria are given time to recover and resume normal growth, with the required nutrients provided to meet the auxotrophic demands.
This method preserves the bacteria for long-term storage while ensuring their viability upon rehydration.
The engineered P. putida strain will be introduced into the soil as part of our solution, designed to colonize the roots of olive trees and establish an endosymbiotic relationship before infection by V. dahliae, thereby providing effective prevention. We envision this as a strategic intervention during periods of heightened vulnerability for olive trees. To ensure successful application, we collaborated closely with local olive producers, whose livelihoods are deeply impacted by Verticillium wilt, gathering crucial feedback to identify optimal deployment times for our irrigation formula.
For saplings destined for transplantation into olive groves, our system will be applied in nurseries as a proactive measure to shield young trees from early Verticillium infection, which could otherwise cause rapid fatality. For mature olive groves, our system will be introduced in spring and autumn, the periods identified by farmers as most susceptible to Verticillium outbreaks due to favorable conditions like high temperatures and humidity.
Temperature and Environmental Conditions [1,2]
Spring Conditions:
1) Temperature: As temperatures begin to rise in spring, they create a conducive environment for fungal pathogens. The optimal temperature range for Verticillium growth typically lies between 20°C and 30°C, which coincides with the warming temperatures of spring.
2) Humidity: Increased humidity levels during this season also play a crucial role. Spring rains can raise soil moisture levels, enhancing the chances of fungal spore germination and root infection. The combination of warm soil temperatures and high moisture creates ideal conditions for Verticillium to thrive and infect vulnerable olive trees.
Autumn Conditions:
1) Temperature: In autumn, the temperatures begin to fluctuate as the days shorten, but warm conditions can persist. Temperatures in the early autumn months may still fall within the optimal range for Verticillium, making it a critical time for infection as trees are still actively growing and may be stressed from the summer heat.
2) Soil Moisture: Autumn often brings increased rainfall, contributing to higher soil moisture levels. This moisture is essential for Verticillium spores, which require water for germination and invasion. The cooler nights can also stress the trees, making them more susceptible to disease.
By aligning our solution with these critical periods, we aim to provide effective, targeted protection for olive trees, promoting their long-term health and resilience against Verticillium wilt.
Greece
Greece follows a cautious approach towards the use and cultivation of GMOs, adhering to European Union (EU) regulations but with additional restrictions. Although EU law permits certain GMO crops, Greece has opted for a national ban on the cultivation of GMOs in many regions, reflecting public sentiment and concerns about environmental impacts [11].
European Union
The EU has stringent regulations regarding GMOs, with clear guidelines for their cultivation, production, and market placement. GMO crops undergo rigorous safety assessments for both human health and the environment before approval. However, member states retain the right to enforce national bans on GMO cultivation, even if the EU has approved the crop. The precautionary principle is at the heart of EU policy, ensuring that GMOs are only permitted if they pose no significant risk [12].
Global Overview
Globally, GMO regulations vary significantly. Countries like the United States and Canada have more relaxed policies, promoting the development and use of GMOs. Conversely, many nations in Africa and Asia have more restrictive frameworks, with some allowing GMO cultivation under specific conditions, while others, like India and China, enforce tighter controls [11].
Before deciding to apply our system in open fields we had to consider the safety of such implementation. The release of a GMO in the environment can be catastrophic for biodiversity and can influence whole ecosystems. Our Wet and Dry Lab departments collaborated once again, each taking a distinct approach to ensure our system is as safe as possible.
From the Wet Lab perspective, we firstly placed many components of our system in the bacterial chromosome in order to prevent horizontal gene transfer. You can see the whole system in our Project Design page. Secondly, we incorporated in our design two different biocontainment methods. We designed a gate that allows the bacteria to grow only in the presence of V. dahliae and introduced a double auxotrophy for L-tyrosine and vitamin B12, two compounds that are scarce in soil environments [13,14]. Additionally, cobalamin being water-soluble, makes it well-suited for inclusion in an irrigation formula [15]. This way the bacteria won’t be able to proliferate in the absence of the pathogen and will die without the external supply of L-tyrosine and vitamin B12.
In the Dry Lab department we aimed to have a more holistic approach of the safety of our system, so we turned our attention to what we can develop in order to help Wet Lab to test biocontainment methods. We created a model that simulates the activity of our engineered bacteria to the soil under different conditions, allowing us to predict potential risks. Moreover, we developed a hardware device that can test biocontainment approaches in situ and monitor bacteria-bacteria interactions. This will help with estimating the effectiveness of different biocontainment strategies, giving us the chance of better optimization.
There are always risks to releasing a GMO to open fields, however we are confident that with a interdisciplinary, SynBio approach and continued optimization of biocontainment methods, will minimize any negative impacts and our system will be safe for implementation.
Determining how to implement our system in the real world required us to address two key questions: 1) “What is the best way to ensure the bacteria will reach the roots?” and 2) “Is this approach realistic and applicable?” To address the first, we consulted literature and experts, including Dr. Athanasios Dalakouras and Professor Aliki Tzima, who both agreed that since the fungus attacks through the roots, delivering the bacteria via an irrigation formula would be the most effective and feasible solution. To evaluate the practicality of this method, we engaged with olive producers, the primary users of the product. While some raised concerns about the cost, particularly for large areas or sloped fields, they expressed a strong willingness to try this method given the severity and long-term impact of the problem, signaling that the approach is both practical and acceptable to the target audience.
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