Team:Wageningen UR/overview/solution

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Wageningen UR iGEM 2014

 

 

Solution


The iGEM team Wageningen 2014 would like to present BananaGuard, a bacterial platform designed to control the Panama disease threatening banana cultivars all over the world. A combination of strategies will prevent Fusarium oxysporum f. sp. cubense from infecting banana plants. Detection of F.oxysporum presence in the soil, by sensing fusaric acid, forms the first part of the BananaGuard system. Expression of four fungal growth inhibitors coupled to the sensor for fusaric acid forms the second part of the BananaGuard system. Making the expression of the fungal growth inhibitors dependent on fusaric acid sensing will enable BananaGuard to have a low impact on non-target organisms. Resistance to fusaric acid is very important in this context. Even though moderate fusaric acid resistance is endogenous for our host organism Pseudomonas putida [1], fusaric acid resistance is engineered allowing the system to be transferable to different host organisms.

When releasing a genetically modified organism into the soil, horizontal gene transfer as well as the risk that non-target organisms are outcompeted need to be taken into account [2]. Therefore biological containment forms the third part of the BananaGuard system. Horizontal gene transfer will be avoided by a double dependent plasmid system. The loss or gain of one of the two modified plasmids will cause the host to die. Additionally, a self-destruction system called Kill-Switch, that will be automatically activated upon BananaGuard completing its function will protect the natural balance within the soil (Figure 1). Engineering a bacterial platform for biological control of F.oxysporum based on P. putida shows great potential. This soil-borne bacterium possesses already a moderate fusaric acid resistance [1] and naturally inhibits F.oxysporum to a certain degree [3].

Figure 1: The BananaGuard time line. After application, BananaGuard will detect F.oxysporum based of fusaric acid secretion, produce fungal growth inhibitors and destroy itself when F.oxysporum can not be detected anymore.

Application

The control of soil-borne pathogens by biological control is not a new idea and has been studied since the early twentieth-century [4]. For a long time commercial applications were thought to be not feasible but, in the last decades techniques for application progressed tremendously.

For BananaGuard, we believe that distributing the final product via drip irrigation is the best method. In drip irrigation a network of pipes is used that has emission points (drippers) delivering the precisely measured volumes of water into the rhizosphere. Distribution of growth substances, such as fertilizers, chemicals, herbicides and fungicides, have been shown to be successful before [5, 6]. In 2008, Boari et al.[7] showed that distribution of suspensions containing small particles via drip irrigation shows great potential. When considering shelf live and transport as well as the fact that, in some growing regions, no mechanical irrigation is used, BananaGuard could be applied as dry products such as granules or powders as seen with comparable products [8]. Furthermore, these dry pellets can be stored for a longer time period. Since BananaGuard is applied as preventative measure, usage of more dilute suspension cultures applied over a longer time period or repeated application of the dry product is conceivable.

Application of BananaGuard could possibly be combined alongside the application of products such as fertilizer. Since farmers, generally, already supplement their plants with fertilizer or pesticides, this would only add a small extra step to their current practices. The farmers can also grow the bacteria themselves with a minimal amount of materials and training, since P. putida is an easily grown bacteria as long as there is the possibility to work in sterile conditions. The bacteria can be harvested over a series of generations. However, new samples would need to be purchased to assure the quality of the product and to remove contamination that arise during harvesting.

Although the application would be simple, regulations concerning an engineered soil bacterium for bio-control are complex and different in most countries. With the current regulations in Europe, for example, application of BananaGuard would not be possible in the near future. Luckily, however, our main target area, South America and Asia have far less stringent rules about the use of GMO products. Combining the acceptance of GMO bacteria in most banana producing countries and maintaining availability of a “GMO free” banana makes our product a strong candidate for introduction into the global market.

As inter-species diversity in commercially-grown crops decreases resistance to pathogens will become weaker. Even though it will not be as dramatic as is the case with bananas, crops will become less diverse and therefore weaker against threats. Fusarium ssp. has been known to also infect other crops such as wheat, cucumbers and tomatoes. If successful, our system could be rapidly changed to work with other crops or even other fungi. By combining the modularity and rapid modification time possible in bacteria but not plants, our system can be an efficient way to safeguard the crops of the future.


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References

  1. Utsumi R, Yagi T, Katayama S, Katsuragi K, Tachibana K, Toyoda H, Ouchi S, Obata K, Shibano Y, Noda M. (1991) Molecular cloning and characterization of the fusaric acid-resistance gene from Pseudomonas cepacia. Agric Biol Chem. Jul;55(7):1913-8.
  2. Wright O., Stan GB., Ellis T. (2013) Building-in biosafety for synthetic biology. Microbiology (2013), 159, 1221–1235
  3. Lemanceau P. Alabouvette C. (1991) Biological control of fusarium diseases by fluorescent Pseudomonas and non-pathogenic Fusarium. Crop Protection. Vol. 10 Issue 4: 279–286
  4. Cook, R. J. (1993). Making greater use of introduced microorganisms for biological control of plant pathogens. Annual review of phytopathology, 31(1), 53-80.
  5. Hebbar SS, Ramachandrappa BK, Nanjappa HV, Prabhakar M (2004) Studies on NPK drip fertigation in Weld grown tomato (Lycopersicon esculentum Mill.). Eur J Agron 21:117–127
  6. Gollehon N (1990) Chemigation: a technology for the future? Economic Research Service, Washington
  7. Boari, A., Zuccari, D., & Vurro, M. (2008). ‘Microbigation’: delivery of biological control agents through drip irrigation systems. Irrigation Science, 26(2), 101-107.
  8. Schisler, D. A., Slininger, P. J., Behle, R. W. & Jackson, M. A. (2004) Formulation of Bacillus spp. for biological control of plant diseases. Phytopathology 94, 1267–1271.