Human Practices
This year, our team was inspired by the agricultural legacy of Texas and concerns about sustainable growing and food security in the era of climate change to investigate solutions to crop loss through synthetic biology. During our ideation process, our team discovered the devastating impacts of bacterial plant pathogens like fire blight and Pierce’s disease, which can destroy entire crops and each contribute to more than $100 million in annual losses in the United States (Giménez-Romero et. al, 2022; Wallis, 2020). Such crop loss also contributes to food insecurity, at a time when natural resources are already strained due to climate change (Singh et al., 2023). Current popular treatments for bacterial pathogens in the United States are also a source of concern, since antibiotics and copper treatments may cause unintended microbial resistance or otherwise harm the surrounding environment (Vu & Oh, 2020).
As we delved into the current research and implications around bacterial pathogens, our team discovered the novel research area of microcins, which have been used by a team at the University of California, Irvine to target specific pathogens without harming nearby microbiota (Vu & Oh, 2020). We envisioned a microcin solution to bacterial plant pathogens, which builds on the sustainable approach of methods like biocontrols by offering enhanced target specificity and a modular system that could be applied to a broad range of pathogens. Acknowledging the need for targeted, sustainable solutions to bacterial pathogens, our team decided to develop a microcin system to protect our state vegetable, the onion, from the bacterial pathogen Pantoea.
Figure 1. Examples of bacterial pathogens affecting crops. Left: grapes impacted by Pierce’s Disease (Westover, 2019); Top right: an apple tree infected with fire blight (Sabins, 2016); Bottom right: an onion with center rot caused by infection with Pantoea ananatis (Schwartz, 2019) , created with BioRender.com.
While developing our microcin system, it was essential for our team to gather insight from stakeholders in our project to ensure it was environmentally conscious and well-catered to the needs of our community. To learn more about the day-to-day problems facing agricultural production, we consulted with growers, sellers, and buyers of produce. Next, our team met with representatives from national and international agricultural initiatives to learn more about the implementation and climate surrounding current broad-scale solutions to bacterial plant pathogens. Meanwhile, we delved deeper into the current research on categorizing and treating bacterial pathogens through our meetings with plant pathologists and microcin experts alike. After each meeting, our team considered the implications of stakeholder feedback for our project design, and we worked in an iterative fashion to integrate this feedback into our wet lab approach and project goals.
Approaching the research, our team encountered many questions and issues vis-á-vis our project outline and design providing the opportunity to reach out to and adjust our planning and goals. This is where our Human Practices faction made serious headway, shifting the focus of our project during its own unfolding.
Figure 2. Human Practices Correspondence Cycle, created with BioRender.com.
Figure 3. Stakeholders, created with BioRender.com.
The Human Practices team met with experts in the fields of plant pathology and plant genetics to refine our wet lab protocols and consider the potential benefits and detriments of using microcins to combat agricultural pathogens like Pantoea. From these meetings, we gained several iterations on how to improve our Onion Center Rot and Red Onion Necrosis assays and suggestions of potential future chassis for our microcin system.
Discussion with Jennifer Parker, Plant Pathologist and Microcin Expert
Figure 4. Jennifer Parker, created with BioRender.com.
During our iGEM team’s ideation process, the Human Practices team met with Jennifer Parker, a Research Scientist in the Davies Lab at the University of Texas at Austin, to discuss bacterial plant diseases of interest and methods used to combat and characterize these pathogens. In this meeting, we discussed the potential of microcin systems to combat common bacterial pathogens such as Xanthomonas, Pseudomonas, and Erwinia, and it became clear that a microcin product could be a promising solution to various agricultural plights being faced by local Texas farmers. As we delved further into the study and application of microcins, our microcin team continued to consult with Jennifer given her expertise in the novel area of research.
Over time, Jennifer became an integral member of the iGEM team, and her role evolved into that of an additional expert advisor for our project. Following our discussion with Dr. du Toit about current biocontrol methods used against Pantoea, the Human Practices team once again consulted Jennifer on the real world applications of microcins. Specifically, we discussed the feasibility of using common biocontrol Bacillus as a potential future chassis for our microcin system, which would allow farmers to smoothly transition from the application of current products to ours.
Discussion with Dr. Brenda Schroeder - Plant Pathologist
Figure 5. Dr. Brenda Schroeder, created with BioRender.com
Our meeting with Dr. Schroeder, a plant pathologist from the University of Idaho, was focused on assessing the viability of our wet lab approach, with a particular emphasis on assays. Key points of discussion included improvements to the Onion Center Rot Assay, such as modifications in temperature, duration, and inoculation methods/concentrations to enhance the assay results. We discussed our conditions, which included incubation at 25°C and using a toothpick for inoculation, and Dr. Schroeder recommended using conditions of 30-35°C and an OD of 0.3, with inoculation using 0.5 mL in a syringe. We also learned how to standardize onion samples at 0.3 OD to achieve a concentration of 10^8 CFU/mL before inoculation. Furthermore, we began conducting cultures on agar plates to ensure culture purity and incorporated water controls and non-inoculated controls into our experiments. We also discussed the onion harvesting timeline and process and explored the potential of utilizing current bacteriocins. Dr. Schroeder was supportive of our project, describing it as a "neat idea" for combating bacterial plant pathogens.
Later on, as our team focussed on finalizing our microcin system, our assays team reconnected with Dr. Schroeder for further insight on how to test our microcins on onions themselves, as a proof of concept for a potential microcin product. In this follow up, Dr. Schroeder helped to clarify details about the process of Pantoea infecting an onion plant, such as how infection usually occurs in the fields rather than during harvest, and how best to employ microcins in the field to prevent infection from Pantoea. Dr. Schroeder proposed that we consider a leaf treatment that could be present on the plant in the event that the plant is wounded, and thus be at the wound site to kill any Pantoea that may enter.
Discussion with Dr. Lindsey du Toit - Plant Pathologist and Stop the Rot Coordinator
Figure 6. Meeting with Dr. Lindsey du Toit
Our conversation with Dr. Lindsey du Toit, a plant pathologist and coordinator for the "Stop the Rot" program, had a dual focus of investigating the institutional solutions to onion rot in the U.S. and the viability of practical applications of our microcin solution. Dr. du Toit also offeredsuggestions for improving our Red Onion Necrosis Assay, including the need for increased moisture by adding wet paper towels, conducting the assay in Tupperware bins, extending the duration from 3 to 5 days, and raising the temperature from 25°C to 30°C. We also considered the bacterial species used in Lifegard (Bacillus mycoides) and its potential viability as a chassis for our microcin system, which we later explored further with Jennifer Parker. Our discussion delved into the onion cultivation process, and we discussed how Pantoea typically infects onions in the field, but may not be identified without expensive X-ray machines that are economically unfeasible for small farms. Thus, we considered how our microcin solution could be catered to the needs of smaller scale farmers, as a preventative measure to offset the advantage of expensive equipment for combating bacterial pathogens. Finally, we established a connection with Subas Malla, a key contact for the Texas "Stop the Rot" initiative.
Finally, our team organized meetings with agricultural initiative and policy representatives to explore how our microcin solution aligns with current initiatives to control plant pathogens nationally and globally, and to discuss how our solution may be received by the greater agriculture market due to policy guidelines. In these meetings, we considered how apprehension to GMOs may pose legislative barriers to developing our solution, and we explored additional local pathogens that could be targeted by future iterations of our microcin system to cater to the changing needs of our community.
Discussion with Dr. Subas Malla - Plant Geneticist and Stop the Rot contributor
Figure 7. Meeting with Dr. Subas Malla
Our conversation with Dr. Subas Malla focused on the local Texas onion rot issue and the coordination of positive and negative controls of Pantoea for further characterization of the pathogen using our Red Onion Necrosis Assay and Onion Center Rot Assay. We also discussed local methods for controlling bacterial pathogens on onions, which commonly include copper treatments and bio controls such as pseudomonas. However, the use of copper in agriculture has been shown to lead to soil pollution, while our microcin system could serve to build on the legacy of using biocontrols as a sustainable alternative to these treatments (Coelho et al., 2020). We also considered the modularity of our system, and we made plans to use our microcin identification pathway to explore potential microcins to combat Burkholderia, another prominent bacterial pathogen in onions. We discussed the potential interest from growers if our solution is established in the field without harming the plants. Agreements were made to send positive and negative control strains of Pantoea, with plans for follow-up.
Discussion with Dr. Mercedes Roca - Agricultural Biotechnology Policy Expert and Plant Pathologist
Figure 8. Dr. Mercedes Roca, Created with BioRender.com.
Our conversation with Dr. Mercedes Roca, an expert in agricultural biotechnology policy and plant pathology, centered on examining the global and historical context for our project, and potential barriers that may exist when considering practical implementation of a microcin system. We discussed the environmental destruction and destabilization caused by climate change and its implications for global food security. We acknowledged Texas' history of agricultural advancements, including the work of Norman Borlaug at Texas A&M, while also considering the consequences of other advancements in the Green Revolution, which increased agricultural productivity but led to excessive fertilizer and pesticide use. We underscored the concept of One Health, emphasizing the interconnectedness of the environment, animal and agricultural health, and human well-being, and we considered how the sustainability of our project may contribute to this ideal. Overall, this conversation highlighted the potential for our project to combat a variety of contemporary agricultural issues, such as food production, bacterial pathogens, and the environmental consequences of current chemical methods for controlling bacterial pathogens.
In this meeting, we also touched upon the misconception that many people, particularly in urban areas of the United States, take food production for granted and often misunderstand the role of GMOs in agriculture. According to Dr. Roca, this misconception has manifested in the form of opposition to genetically modified organisms (GMOs), with “activists” mistakenly framing the shortcomings of agriculture as primarily one of distribution rather than production. This misperception is especially threatening in a state like Texas, where agriculture comprises a majority of the land in Texas (74% of the state’s 268,581 square miles) and a large portion of the population’s career involvement as 14% of working Texans are directly implicated in an agricultural-related job (Hundl, 2019;” Texas Ag Stats,” 2023). To further investigate the climate and potential pushback around GMOs in our community, our team visited the local Mueller Farmers’ Market and engaged buyers and sellers of produce in conversations about our project and GMOs, which is described below.
In our follow up discussions with Dr. Roca, we were encouraged to go into greater detail when communicating our microcin system to stakeholders, which we were able to implement in our later visit to the farmers’ market. We were also informed that antibiotics are not commonly used for global agricultural practices, and thus farmers in other countries may have the same concerns of antibiotic resistance as farmers in the US when using traditional methods to combat bacterial pathogens in agriculture. However, these farmers may still find our solution relevant as an alternative to chemical treatments against bacterial pathogens, and we found that our proposed practical application of microcin-containing spray aligned with global agricultural practices of spraying treatments on crops.
Figure 9. Texas’ Agricultural Land Distribution, created with BioRender.com.
Finally, our Human Practices team engaged with local Texas farmers to discuss the applicability of our proposed solution to their current cultivation practices, and consider ways in which we could make the implementation of our microcin system more practical for farm life. We also engaged in conversations with buyers and sellers of agricultural produce to gauge the general attitude of our community toward synthetic biology products. From these discussions, our team was inspired to envision a spray application of our future microcin system, and we were reminded of the importance of effective science communication in gaining support for our solution.
Discussion with Rising Sun Vineyard - Local Vineyard
Figure 10. Stakeholder meeting with farmers at Rising Sun Vineyard. Taken by Elizabeth Manriquez
Figure 11. Meeting with Shayna, Rising Sun Vineyard
Our initial discussion with Shayna, a representative from local Rising Sun Vineyard, revolved around assessing the viability of our microcin system for Texas agriculture. We discussed current issues facing vineyards and small scale agricultural producers in general, including weather damage, climate concerns, fungal diseases, and mold. Vineyards like Rising Sun also combat bacterial pathogens, such as Xyllela, which can be spread through sharpshooter infestations and destroy entire crops in a matter of days. We talked about the current methods used to deal with Xylella and sharpshooter infestations, which often involve antibiotics administered through the water source or the use of resistant varietals, although the flavor of these American varietals is often considered inferior to European varietals.
After this initial meeting, we arranged for our team to make an excursion to Rising Sun Vineyard to see the production and protection of agriculture in practice, and we met owner Steve Frintz. During this excursion, we further discussed the viability of a microcin product in the eyes of the Rising Sun representatives, and they expressed interest in a potential microcin product that could be applied through a spray or drip to mimic currently available treatments for bacterial pathogens. The representatives also conveyed the broader interest of their vigneron associates, indicating that their associates at other vineyards would also likely be interested in a microcin treatment, given the other vineyards' plight of Xylella. Overall, although our project could not address all problems faced by the growers at Rising Sun Vineyard, our visit established the potential for a microcin solution to lessen the burden experienced by growers as they adapt to an unpredictable environment.
Discussion with Buyers and Sellers of Local Produce, Mueller Farmers’ Market Image
Figure 12. Buyers and Sellers of Agricultural Produce, created with BioRender.com.
To gauge public perception of our strategy for tackling bacterial pathogens in agriculture, our team visited the Texas Farmers’ Market at Mueller and interviewed buyers and sellers of local produce. Prior to this visit, we considered the shifting perception of GMOs in the United States, in which recent legislative changes now require the labeling of GMO products (“How GMOs are Regulated”, 2023). Given these recent changes, our team was interested in how public perception of GMOs might affect the community’s openness to our microcin solution, and we used a brief discussion about GMOs to contextualize our conversations with farmers’ market attendees.
Our discussion with one local seller of organic produce highlighted the potential skepticism our solution might face due to being labeled as a GMO or GM biocontrol product. Specifically, this seller was concerned about the consequences of our product, and of all GMOs, on human health, and she expressed that she held a “poor” perception of GMOs in general. However, given the clarification that our solution would not involve genetically modifying the crop itself, and instead would only modify a product that could be applied and washed off of the crop, the seller found our idea “interesting.” Finally, the seller expressed concern about the application process of our product adding time to farmers’ long days, but maintained that she would be interested to learn more about our project if provided with educational materials such as a website.
Meanwhile, our conversations with buyers of local produce offered a more promising outlook on the public perception of our microcin solution. Buyers seemed familiar with the issue of bacterial resistance, and were open to potential GMO alternatives given the adverse environmental effects of bactericides and pesticides. As one buyer acknowledged, the big hurdle for our project would be overcoming the public perception of GMOs in our attempt to explain and advertise a microcin product. In the eyes of another buyer, the common practice of “lumping anything that's genetically modified into one category and saying we aren't going to do that is too extreme,” but it is still important to consider the risks of implementing a GM product.
Overall, our conversations with buyers and sellers of produce at the Texas Farmers’ Market at Mueller highlighted the importance of clearly communicating our microcin system to stakeholders, and we continuously refined our presentation of our microcin solution throughout these conversations. Additionally, we were reminded to cater our considerations of future practical applications for our microcin solutions to the day-to-day need of farmers, which re-emphasized the viability of spray or drip methods discussed with Rising Sun Vineyard.
Overall, the work of our Human Practices team was key for gaining feedback on both our wet lab methods and project approach, and we were able to reflect on stakeholder feedback throughout our project’s progression to adjust our proposed solution to the needs of our community. From these conversations, we were left with three key takeaways regarding the practical application of microcins as a solution to bacterial plant pathogens.
- A practical application of our project should utilize the modularity of microcins to address bacterial pathogens that could be most impacted by our solution, such as those causing the greatest concern to local farmers despite currently available treatments.
While our conversation with Dr. du Toit indicated that Pantoea is a source of concern for farmers in the United States, our meetings with Dr. Malla and representatives from Rising Sun Vineyard indicated that Texas farmers may be more concerned about the threat of pathogens like Bulkhoderia and Xylella given our state’s particular climate. Although Pantoea provides a helpful model for testing our microcin system, further development of our project into a practical product should use our microcin identification pathway to adjust our microcin system to address these more pressing pathogens.
- Any future microcin product should be accessible to farmers, and should mimic currently used techniques, like sprays or irrigation, for easy application.
Our conversations with Dr. Malla and sellers of agricultural produce indicated that one of the largest barriers for implementing our proposed solution would be the potential inconvenience our method may impose on the farmers. Therefore, it is essential that a practical implementation of our project mimics the application methods of current treatments so that our solution is feasible for busy farmers.
- Promotion of a microcin product should be educational, well labeled, and with resources available for farmers, sellers, buyers, academics, and the like.
In our conversations with Dr. Roca, we were reminded that microcins are a nascent area of research, and many experts in the field of plant pathology have yet to hear about their mechanisms and practical applications. Additionally, our discussions with buyers and sellers emphasized the importance of science education for addressing misconceptions about microcins and GMO products in general. Any plans to promote a microcin product must prioritize educational labeling and thorough explanations of how microcins work to prevent misguided apprehension toward our solution.
While our conversation with Dr. du Toit indicated that Pantoea is a source of concern for farmers in the United States, our meetings with Dr. Malla and representatives from Rising Sun Vineyard indicated that Texas farmers may be more concerned about the threat of pathogens like Bulkhoderia and Xylella given our state’s particular climate. Although Pantoea provides a helpful model for testing our microcin system, further development of our project into a practical product should use our microcin identification pathway to adjust our microcin system to address these more pressing pathogens.
Our conversations with Dr. Malla and sellers of agricultural produce indicated that one of the largest barriers for implementing our proposed solution would be the potential inconvenience our method may impose on the farmers. Therefore, it is essential that a practical implementation of our project mimics the application methods of current treatments so that our solution is feasible for busy farmers.
In our conversations with Dr. Roca, we were reminded that microcins are a nascent area of research, and many experts in the field of plant pathology have yet to hear about their mechanisms and practical applications. Additionally, our discussions with buyers and sellers emphasized the importance of science education for addressing misconceptions about microcins and GMO products in general. Any plans to promote a microcin product must prioritize educational labeling and thorough explanations of how microcins work to prevent misguided apprehension toward our solution.