Team:Tec-Monterrey/Description

DESCRIPTION

The importance of agave

Agave is a crucial part of Mexico’s culture, ethnicity, and economy. In our country, Agave is produced in over 550 municipalities which represents almost a quarter of the nation’s total [1], its high demand is due to the manufacture of alcoholic beverages such as mezcal, bacanora, pulque and of course, tequila. This last one is especially important for Mexico’s economy, since it generates a net income of more than 1 billion USD every year [1]. Tequila is obtained by the distillation of the sugars contained in agave, specifically from the heart or “piña” of Agave tequilana. The “piña'' is also known as “mezcal” (yes! like the beverage) which in the nahuatl dialect means “la casa de la luna” or “the house of the moon” [2]. Tequila is considered to be the Mexican drink of choice, but its production faces an obstacle: the wilting of agave.

The problem

Wilting of agave is caused by a diversity of fungi. It has also been known as “tristeza” (sadness) of agave, since it causes the irreversible necrosis of the infected plant, which makes the leaves look as if they were sad and leads to the eventual death of the agave [3]. Among the fungi causing this disease is the genus Fusarium spp., one of the main species being Fusarium oxysporum which can also infect a variety of other plants by attacking their vascular system. In Mexico it has been reported that between 60% and 100% of a crop can be lost due to the late diagnosis of wilting of agave [4]. This fungus also produces a variety of mycotoxins harmful for humans, such as nivalenol and T-2 toxin [5]. The former produces cellular apoptosis, specifically in blood cells which are sensitive to mycotoxin exposure, and the latter induces haemorrhages and necrosis in the GI tract, reproductive organs and hematopoietic organs such as the bone marrow [6][7].

Currently, there is no cure for the wilting disease, meaning that the best thing to do is to diagnose it at an early stage and isolate or dispose of the infected plants [8].

Current solutions

Current detection systems are either visual or laboratory examinations. There is the “Muestreo en Cinco de Oros” technique, which divides the area in five in order to evaluate plant’s necrosis on a scale from 0 to 2 according to its severity [9], but since it is a visual analysis it may be too late for when it’s executed. Laboratory testing includes ELISA, PCR or direct observation and identification of the fungi under the microscope. The disadvantage of these examinations is the time that it takes the results to get back to the farmers, since the sampling is done by a specialized person traveling from miles and miles away. Overall, the process can take up to three weeks, and by that time the infection can be spread through the whole land, causing the total loss of the agave crops and making the farm a source of infection [9].

Our solution

As we analyzed this problem, we came to the conclusion that our solution should be able to generate the results in a short period of time and require minimal specialization of the user. After reviewing many past igem projects (such as ViTest by EPFL 2019 and Rosewood by Evry_Paris-Saclay 2020), as well as scientific articles addressing similar issues [10][11][12], we came up with our final proposal: Diagnosgene.

Diagnosgene consists of a rapid, easy-to-use, and safe early detection system which can be implemented on the field generating results of the diagnosis within hours. The detection of Fusarium oxysporum is accomplished by the usage of toehold switches, which are RNA tools that get specifically attached to a given complementary sequence, leading to the expression of a reporter gene.

See Design > Detection to learn more about toeholds.

As a crucial part of the project, we developed a software to standardize the creation of these switches, since the process to design and analyze the necessary sequences isn’t trivialand it normally takes more than one platform to do so, tending to be very confusing for someone who is not specialized in the subject; so we came up with the user friendly Toehold Switch Creator, which facilitates both the design and analysis of these genetic tools not only for phytopathogenic detection, but for its use in many fields.

You can visit the Software page to read all about it.

Lastly, we agreed that we did not want a biosensor on a living organism, since our platform is thought to be utilized on the field and it could lead to its accidental release to the environment and become harmful to the farmers and ecosystem. Taking this into consideration, we decided to develop a cell-free system, which will carry out the reactions and which we engineered to be low-cost by substituting expensive reagents traditionally used since we want our product to be accessible.

Find out more about our cell-free on the Design > Cell-free page

DESIGN

What are you waiting for? Read the technical details of our project at our Design page.

 HUMAN

Check out how we worked side by side with farmers and experts at our Human Practices page.

References

  1. Vázquez-Elorza, A. (October 2016). El agave en México, temática socioeconómica. INVDES. https://invdes.com.mx/los-investigadores/el-agave-en-mexico-tematica-socioeconomica/
  2. Consejo Regulador del Tequila (2019). Historia. https://www.crt.org.mx/index.php/es/el-tequila-3/historia
  3. Fucikovsky L. (2001) “Tristeza” and Death of Agave tequilana Weber var. Blue. In: De Boer S.H. (eds) Plant Pathogenic Bacteria. Springer, Dordrecht. https://doi.org/10.1007/978-94-010-0003-1_81
  4. Villa-Martínez, A., Pérez-Leal, R., Morales-Morales, H., Basurto-Sotelo, M., Soto-Parra, J. M., & Martínez-Escudero, E. (2015). Situación actual en el control de Fusarium spp. y evaluación de la actividad antifúngica de extractos vegetales. Acta Agronómica, 64(2), 194-205. https://dx.doi.org/10.15446/acag.v64n2.43358
  5. Gómez-Ayala, A. M. (2007). Micotoxinas en alimentos. Farmacia profesional, 21(8), 49-53. https://www.elsevier.es/es-revista-farmacia-profesional-3-articulo-alimentos-micotoxinas-13109791
  6. Minervini, F., Fornelli, F., & Flynn, K. M. (2004). Toxicity and apoptosis induced by the mycotoxins nivalenol, deoxynivalenol and fumonisin B1 in a human erythroleukemia cell line. Toxicology in Vitro, 18(1), 21–28. https://doi.org/10.1016/s0887-2333(03)00130-9
  7. Adhikari, M., Negi, B., Kaushik, N., Adhikari, A., Al-Khedhairy, A. A., Kaushik, N. K., & Choi, E. H. (2017). T-2 mycotoxin: toxicological effects and decontamination strategies. Oncotarget, 8(20), 33933–33952. https://doi.org/10.18632/oncotarget.15422
  8. Secretaría de Ganadería, Agricultura, Desarrollo Rural, Pesca y Alimentación. (2018). Estrategia operativa de la campaña contra plagas reglamentadas del agave. https://www.gob.mx/cms/uploads/attachment/file/283583/Estrategia_operativa_2018_agave.pdf
  9. Secretaría de Ganadería, Agricultura, Desarrollo Rural, Pesca y Alimentación. (2017). Manual operativo de la campaña contra plagas reglamentadas del agave. https://www.gob.mx/cms/uploads/attachment/file/625775/Manual_operativo_de_la_campa_a_contra_plagas_reglamentadas_del_agave_compressed__1_.pdf
  10. Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold switches: de-novo-designed regulators of gene expression. Cell, 159(4), 925-939. https://doi.org/10.1016/j.cell.2014.10.002
  11. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W. & Collins, J. J. (2016). Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell, 165(5), 1255-1266. http://dx.doi.org/10.1016/j.cell.2016.04.059
  12. Chakravarthy A, Nandakumar A, George G, Ranganathan S, Umashankar S, Shettigar N, Palakodeti D, Gulyani A, Ramesh A. Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay. Life Sci Alliance. 2021 Sep 30;4(12):e202101213. https://doi.org/10.26508/lsa.202101213