Implementation
Introduction
Throughout iGEM, our approach has been focused on the development of a translational product that tackles the issue of fungal diseases in a matter that is tailored to the end user's needs and context. As outlined in detail in our human practices and integrated human practices pages, this framework has resulted in continuous interactions with our stakeholders, which, in turn, have significantly impacted our implementation plan.
End Users
Our project has the potential to impact various stakeholders, both directly and indirectly by virtue of the numerous industries affected by the cost of staple crop produce. We are invested in increasing the sustainable productivity of smallholder farmers, the engines of economic growth and prosperity for themselves and their country, the men and women who hold the keys to food secure homes. Given the ease of use, storage, and implementation within existing farming practices, we envisage our product being adopted by NGOs and farming cooperatives as a powerful tool to share with smallholder farmers in developing countries and resource-poor areas. A group for whom fungicides and new resistant seeds are often inaccessible, especially in the current market, making them extremely vulnerable to losing significant proportions of their crop to fungal pathogens.
User Centred Design
We made several design choices, In light of who are end users are and the broader context of the issue we are tackling, so as to ensure that we tackling the right problem in the right way.
Why broad spectrum?
As part of an integrated pest management system, most farmers perform crop rotations. Each crop may be vulnerable to different and multiple fungal pathogens that are present in the environment, beyond any single most abundant or devastating strain [1]. In order for a product to be as effective as synthetic fungicides and replace their use, it cannot target a single pathogen, as that would require farmers to use multiple products on a single crop, driving up costs. This is especially relevant when looking at the case of smallholder farmers. Speaking to Dr. Bhavani from the CIMMYT, we learnt the importance of maintaining strong crop protection levels across farms in a region, else resource-poor farmers’ crops can become a breeding ground for fungal pathogenic spores that infect commercial farms and create a high disease pressure scenario.
Furthermore, to ensure efficacy we must take into consideration the role of emerging fungal pathogens. These can be defined as pathogens that “cause a new disease, greatly increase the incidence of a disease or introduce a disease to a new geographical location or infect a new host”. [2] Three main factors have lead to a perfect storm of pathogen emergence, which now poses a significant risk to global food security. The loss of crop diversity worldwide has catalysed the rise of new virulent strains that are capable of overcoming resistance genes bred into seeds [3]. An example is the now redundant resistance gene Yr17 which conferred wheat yellow-rust resistance [4]. Second, the overuse and over-reliance on single-target-site fungicides has led to the rapid emergence of new strains of fungi. Currently, fungicide resistance has developed against all major classes of single-target-site fungicides in several major crop pathogens, such as triazole-resistant Z. tritici affecting UK wheat [5]. Third, due to ecological degradation and climate change, environmental permissiveness towards the rise of new diseases and virulence has increased. Climate factors such as temperature and humidity affect population size, reproduction speed and mutation rates [2]. Researchers have found that increasing temperature may create greater pathogen genetic variation, increasing the ability of fungi such as Z. tritici to evolve novel virulence mechanisms and overcome barriers such as fungicide application [6]. One recent example has been the surging threat of wheat blast to production in Bangladesh, previously foreign to the region, due to both anthropogenic pathogen transport and climate changing to be more conducive to blast disease [7]. This evidence makes it clear that the development of a novel fungicide that is single-target or pathogen specific, would be futile in the fight against fungal pathogens. According to both literature and our interviews with experts such as Dr Oliva Ricardo & Dr Lorena Maxwell, what is needed now are new antifungals with either broad-spectrum antifungal activity or the ability to boost plant defences, as these mechanism have been found to result in the lowest risk for the emergence of fungicide resistance [8]. Our project aims to develop a product that is equipped with both these qualities.
Why Chitin?
As highlighted above, researchers have found optimal biocontrol strategies to have either of two modes of action: broad-spectrum antifungal activity and plant immune response upregulation. Our goal was to engineer a system that could do both. This lead us to targeting chitin as a molecule of interest.
Chitin is a linear homopolymer of β-(1,4)-linked N-acetyl-D-glucosamine (NAG) monomers. It is one of the major components of fungal cell walls, forming a network with glycoproteins to provide structure and stiffness. In pathogenic fungi, the cell wall plays a key role in host invasion, as the first structure to make contact with host cells. In response, plants have developed mechanisms to detect several components of this layer, in order to activate local and systemic immune responses [9]. The recognition of chitin monomers plays a fundamental role in the establishment of basal resistance to potential pathogens in plants. Chitin is in fact, a well-known general elicitor of plant innate immunity [10]. Since the 1980s, chitin has been used in pest management to prime plants, such that they exhibit a greater resistance against infections via a phenomenon called systemic acquired resistance (SAR) [11] [12]. Chitinase enzymes have also been explored as a supplement to commonly used fungicides, not only to increase their potency by inhibiting the growth of pathogenic fungi, but also to reduce the concentration of synthetic active ingredients which have been found to be harmful to both human health and the environment [13].
Why Bacillus spores?
In one sentence, B. subtilis spores are cheap, durable, robust protein display systems that would provide a long protection window. Our goal is to exploit the innate properties of spores so as to develop a product that is equitable, accessible and robust.
Bacillus subtilis is a plant-growth-promoting rhizobacteria, present in the natural soil microbiome that induces resistance to both abiotic and biotic stressors in plants via both direct and indirect modes of action. There is particular interest in B. subtilis as a biocontrol agent, due to its strong antifungal properties and GRAS (generally regarded as safe) status from the FDA, with evidence of being non-pathogenic nor toxicogenic to plants, animals and humans [14]. The bacteria possesses several different mechanisms through which it bolsters defences against fungal pathogens [15]:
Overall, more than 24 antibiotic substances have been reported as being produced by B. subtilis, this included peptides, proteins and non-peptides based substances [16]. Having such an agent, would greatly limit the likelihood of emergence of resistance in pathogens. B. subtilis would not only act as an excellent biocontrol agent, as has been already observed in practice, it would also confer an advantage. Inoculation of a Bacillus strain was found to have a positive effect on root nodulation, enzyme production and plant growth when compared to non-inoculated palants. Additionally, the interaction between AM fungi in the soil and B. subtilis has been found to improve yields, namely inoculation of geranium with both microbes resulted in yield increase by 59.5% (an increase of only 49.4% was observed with AM fungi alone) [17].
Bacteria such as Bacillus subtilis, have evolved multiple strategies to weather extreme environmental conditions. Among these is the ability to form spores, also known as sporulation. Spores are the most dormant form of bacteria, they are partially dehydrated and exhibit minimal respiration, metabolic activity and enzyme production. Bacillus subtilis spores are characterised by a multilayered protective structure (at least 4 distinct ones) composed of 70 different proteins. Thanks to this architecture and its composition, spores are extremely resistant to a variety of environmental extremes, ranging from high temperature, UV radiation and desiccation to pH extremes and toxic chemicals [18]. Upon the detection of appropriate nutrients, such as L-alanine, these spores can rapidly return to a vegetative state through a process called germination. Although the upper limit of their viability is not known, reports in literature document revival of spores from samples that range from being decades to several thousand years old [19].
Interestingly, the significant stress resistance of spores can be exploited to improve the stability and promote the reusability of enzymes, through their immobilisation on the spore coat. Greater thermostability has been observed in surface-displayed enzymes such as lipases. Extensive research into spore coat proteins, also means that researchers have a roster of anchor proteins to choose from when designing a fusion protein for the immobilisation of an enzyme [20]. The use of spores overcomes one of the biggest limitations of bio-based fungicides, whether these be microbial or biochemical: persistence and optimal activity in-field. Due to the vulnerability of biocontrol agents to fluctuation in temperature and humidity, their translation to in field use has been severely restricted, despite their excellent performance in vitro. Adhesins present in the spore coat would also prevent the “washing away” of biocontrol agent, as is the case with the spraying of microbes.
The durability of spores also addresses several other challenges associated with biofungicides: their storage, transport and production. The use of spores thus enables the production of a solution that is equitable, as it should be accessible without additional costs to farmers working in resource-poor areas and remote locations, far from the site of manufacture. Furthermore, efficacy can be assured even with highly variable weather conditions, as is likely to become a norm due to climate change.
Why direct spraying of spores?
Speaking to farmers across Europe as well as India, we quickly learnt the versatility of a spray that is able to fit into current agricultural practices. Most farmers employ spraying as a method for application of both fungicides and pesticides, including weed suppression products [21]. Thus this infrastructure is present or accessible in established farming enterprises, allowing for the application of multiple different products through the same tool, reducing costs on the long-term. Integrating into this existing framework would make our product more like to be adopted by farmers, due to its convenience and low-labour demanded.
Spraying is also preferable when focusing on efficacy. Fungal pathogens has two stages of infection: host invasion and colonisation. Invasion into plant tissues occurs through either natural plant opening such as stomata, or wound sites, such as those caused by insects [22]. Thus the aerial part of plants is most at risk. To efficiently cover this surface area with biocontrol agent, spraying is the most effective application method.
Implementation Plan
Figure 1: Product Roadmap
Smallholder farmers will only require a smaller quantity of our product making them a useful customer whilst the scale of our manufacture is increasing. However, since 70% of the world’s food is produced by industrial scale farms, we will eventually need to transition to large scale farms as our target customer to develop a substantial market share [23]. We will then seek to raise money for large scale manufacturing facilities based on our success with smallholder farms.
We will first seek regulatory approval in the US as many countries follow their lead for food safety and environmental regulations. Thus, the US will act as our beachhead market. Although we have designed our solution to be compliant with European regulations, we anticipate it being more difficult to obtain approval as well as public acceptance of our product in the EU than in other parts of the world. Therefore, we will not aim to sell our product in the EU until we have secured a substantial market share elsewhere. As the largest geographical market for fungicides is in Asia Pacific (at 38.2% of the global market) we will also target our initial efforts to sell the product there [24].
Finally, thanks to the durability and resilience of spores, our biofungicide platform has the ability to be stored for very long periods of time without requiring any costly infrastructures, being able to retain functionality for up to 10 months at room temperature storage. This would contribute in making our solution accessible to developing countries, where fungal diseases are not only associated with profound economical losses for producers but also to severe food supply insecurity for consumers, aligning our project with the “Zero Hunger” UN sustainability goal.
Safety and Challenges
As our cultivation systems face more stress than ever before, it is time for the development of novel and creative solutions to defend our food security. Over the last couple of decades, the public perception of GMO crops has been less than positive. Often stigma surrounding GMOs, alongside negative emotions stemming from both misunderstanding and failure in the past, have made space for dialogue on the topic very restricted. As public debate, understanding and opinion in the matter leans towards the negative side, avenues toward more comprehensive and science-based legislature are blocked. This can curtail much needed innovations, especially in the field of agriculture. Our hope is that by having strong biosafety strategy, where no foreign plasmid DNA is present in the final product, as well as an approach where engineered biology is not consumed, we can invite a fresh perspective on use in agriculture.
Indeed, our project is designed to produce engineered spores that do not contain any foreign DNA: once the system is activated only wildtype bacteria (B. subtilis) would be produced. However, the efficacy of our spores relies on a self-digesting cassette. Should this fail unexpectedly and not be detected prior to release, there is a risk of autonomous spread of engineered organisms. Further development of our project would require in planta and in field testing to understand In regards to in field testing, we envision completing these in the US under the framework developed by the EPA. Given that our project consists of a live organism that presents engineered proteins but no foreign DNA, we believe we would need to account for risks not examined in current regulatory frameworks developed around GMOs.
Our conversations with the public, academics, farmers and legislators have informed an understanding of the real risks our product may pose if released, enabling us to develop a product-based risk assessment to appropriately address concerns and identify potential solutions. We hope that initiatives such as this facilitate the development of a new narrative - one where synthetic biology is employed consciously and responsibly to tackle life-threatening global challenges with an ethos of sustainability and equity at heart.
References
[1] Fisher, M. C. et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484, 186 (2012).
[2] Fones, H.N. et al. (2020) ‘Threats to global food security from emerging fungal and oomycete crop pathogens’, Nature Food, 1(6), pp. 332–342. Available at: https://doi.org/10.1038/s43016-020-0075-0.
[3] Fones, H. N., Fisher, M. C. & Gurr, S. J. Emerging fungal threats to plants and animals challenge agriculture and ecosystem resilience. Microbiol. Spec. https://doi.org/10.1128/microbiolspec.FUNK-0027-2016 (2017).
[4] Bayles, R., Flath, K., Hovmøller, M. and de Vallavieille-Pope, C., 2000. Breakdown of the Yr17 resistance to yellow rust of wheat in northern Europe. Agronomie, 20(7), pp.805-811.
[5] Sagatova, A., Keniya, M., Wilson, R., Sabherwal, M., Tyndall, J. and Monk, B., 2016. Triazole resistance mediated by mutations of a conserved active site tyrosine in fungal lanosterol 14α-demethylase. Scientific Reports, 6(1).
[6] Bernard, F. The Development of a Foliar Fungal Pathogen Does React to Temperature, but to which Temperature? PhD thesis, AgroParisTech (2012)
[7] Mottaleb, K. A. et al. Threat of wheat blast to South Asia’s food security: an ex-ante analysis. PLoS One 13, e0197555 (2018).
[8] Steinberg, G. and Gurr, S., 2020. Fungi, fungicide discovery and global food security. Fungal Genetics and Biology, 144, p.103476.
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[10] Kaku, H. et al. (2006) ‘Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor’, Proceedings of the National Academy of Sciences, 103(29), pp. 11086–11091. Available at: https://doi.org/10.1073/pnas.0508882103.
[11] Takagi, M. et al. (2021) ‘Chitin-induced systemic disease resistance in rice requires both OsCERK1 and OsCEBiP and is mediated via perturbation of cell-wall biogenesis in leave’. Available at: https://doi.org/10.1101/2021.11.30.470685.
[12] Pusztahelyi, T. (2018) ‘Chitin and chitin-related compounds in plant–fungal interactions’, Mycology, 9(3), pp. 189–201. Available at: https://doi.org/10.1080/21501203.2018.1473299.
[13] Nagpure, A., Choudhary, B. and Gupta, R.K. (2014) ‘Chitinases: in agriculture and human healthcare’, Critical Reviews in Biotechnology, 34(3), pp. 215–232. Available at: https://doi.org/10.3109/07388551.2013.790874.
[14] R.M. Martinez, in Brenner's Encyclopedia of Genetics (Second Edition), 2013
[15] Hashem, A., Tabassum, B. and Fathi Abd_Allah, E. (2019) ‘Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress’, Saudi Journal of Biological Sciences, 26(6), pp. 1291–1297. Available at: https://doi.org/10.1016/j.sjbs.2019.05.004.
[16] T. Wang, Y. Liang, M. Wu, Z. Chen, J.Lin, L. Yang Natural products from Bacillus subtiliswith antimicrobial properties, Chin. J. Chem. Eng., 23 (4) (2015), pp. 744-754
[17] D.K. Großkinsky, R. Tafner, M.V.Moreno, S.A. Stenglein, I.E.G. De Salamone, L.M. Nelson, T. Roitsch Cytokinin production by Pseudomonas fluorescens G20–18 determines biocontrol activity against Pseudomonas syringae in Arabidopsis
[18] Hashem, A., Tabassum, B. and Fathi Abd_Allah, E. (2019) ‘Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress’, Saudi Journal of Biological Sciences, 26(6), pp. 1291–1297. Available at: https://doi.org/10.1016/j.sjbs.2019.05.004.
[19] Ulrich, N., Nagler, K., Laue, M., Cockell, C., Setlow, P. and Moeller, R., 2018. Experimental studies addressing the longevity of Bacillus subtilis spores – The first data from a 500-year experiment. PLOS ONE, 13(12), p.e0208425.
[20] Aguilar, C., Vlamakis, H., Losick, R. and Kolter, R., 2007. Thinking about Bacillus subtilis as a multicellular organism. Current Opinion in Microbiology, 10(6), pp.638-643.
[21] Tudi, M., Daniel Ruan, H., Wang, L., Lyu, J., Sadler, R., Connell, D., Chu, C. and Phung, D., 2021. Agriculture Development, Pesticide Application and Its Impact on the Environment. International Journal of Environmental Research and Public Health, 18(3), p.1112.
[22] Plant Disease: Pathogens and Cycles (2016) CropWatch. Available at: https://cropwatch.unl.edu/soybean-management/plant-disease (Accessed: 12 October 2022).
[23] Smallholders produce one-third of the world’s food, less than half of what many headlines claim (no date) Our World in Data. Available at: https://ourworldindata.org/smallholder-food-production (Accessed: 6 June 2022).
[24] Global Fungicides Market Size & Growth Report, 2020-2027. Available at: https://www.grandviewresearch.com/industry-analysis/fungicides-market (Accessed: 6 June 2022).