Team:Tec-Chihuahua/Design

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Design

Overview

Our project's objective is finding an effective treatment for Verticillium dahliae on cotton crops that can replace current agrochemicals. Taking advantage of our community’s closeness with the cotton industry, we decided to reach out to agronomical engineers, cotton producers, investigators, and other experts on the topic to work out the perfect solution. Taking into consideration their feedback and the customer's comfort, a conclusion was reached. Due to Verticillium dahliae's infection mechanisms, our fungicide must be both a preventive and treating product that could be applied through the irrigation system.

In order to fulfill this goal, through several days of research, brainstorming, and troubleshooting, we developed the design of V-TION: a systemic, preventive, and treating biopesticide against Verticillium wilt with three recombinant plant defense peptides as its active ingredient.

How We Chose Our Peptides

The inspiration for considering peptides as our active ingredient came from the recent interest in their usage as an alternative to current pesticides and antibiotics. Since antimicrobial peptides are effective against various pathogens including fungi, they have been hypothesized as a possible replacement to pest-control chemicals that can be hazardous for the environment and the consumer.1

In order to make an optimal biopesticide, we chose 3 specific peptides from plant's innate defense systems:

  • Arabidopsis thaliana profilin 1 (AtPFN1).
  • Pinus sylvestris defensin 1 (PsDef1).
  • Wheat antimicrobial peptide 1b (WAMP1b).

They were chosen because each one counts with a different action mechanism. When used together in our fungicide, they can work collaboratively to generate an overall stronger antifungal effect that will inhibit V. dahliae at different stages of its life cycle.

AtPFN1

The first stage of V. dahliae’s life cycle is the dormant phase. During this period the fungus survives as microsclerotia in soil during unfavorable conditions. The microsclerotia germinate in the presence of root exudates. Eventually, it enters and spreads through the roots by mycelium or spores.2 Thanks to its ability to permeabilize the cell wall and membrane of fungal spores and hyphae, AtPFN1 can act during this early stage.3

PsDef1

During the parasitic stage, the fungus invades the plant, and its tissues become necrotic because the vascular tissue is clogged by mycelia and conidia.4 PsDef1 is able to halt fungal growth by causing morphological changes on the mycelium.5 It was chosen due to its functionality against mycelium and its potential during this stage.

WAMP1b

When afflicted by fungal disease, plants can fight back by producing chitinases, enzymes that bind and cleave hyphal chitin. Then, some fungi, like V. dahliae, produce fungalysin, a metalloprotease that specifically cleaves and inactivates chitinases, suppressing its function. To counteract this, plants like wheat express WAMPs that bind to fungalysin and inhibit its activity. This way, chitinases remain active to suppress the effect of the pathogen.6,7 Our biofungicide will include WAMP1b among its active ingredients to imitate this system and suppress V. dahliae's defense mechanisms.

Genetic Design

Once we had chosen our peptides, important considerations were taken to build the perfect DNA construct that would allow us to express them recombinantly. Escherichia coli was chosen as an expression chassis for its availability and growth simplicities. However, two of our peptides, PsDef1 and WAMP1b, contain multiple disulfide bonds which are modifications hard to replicate in prokaryotic expression systems. Disulfide bonds are involved in the structural and catalytic activity of proteins; besides, misfolding of these bonds can cause the formation of aggregation bodies and low yields in protein production.8 Our BioBricks were designed to foment the correct folding of these peptides in the right chassis.

Chassis

E. coli BL21 (DE3)

This strain was chosen as a chassis for the expression of AtPFN1. The DE3 designation means that respective strains contain the λDE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter. IPTG is required to maximally induce expression of the T7 RNA polymerase in order to express recombinant genes cloned downstream of a T7 promoter. BL21(DE3) is suitable for expression from a T7 or T7-lac promoter.9 BL21(DE3) strains lack the expression of Lon and OmpT proteases which generates conditions more favorable for recombinant protein production.10

E. coli SHuffle

The cytoplasm of wild-type E. coli is an unfavorable environment for the production of disulfide-bonded proteins due to the presence of reductases and reducing agents. To solve this issue, E. coli SHuffle strains lack two reducing pathways and expresses DsbC, a disulfide bond isomerase, in the cytoplasm to fix improperly folded disulfide-bonds containing proteins.11 For the expression of both PsDef1 and WAMP1b, E. coli Shuffle T7 Express from New England BioLabs was chosen as our chassis.

Promoters

T7 Promoter

The usage of inducible promoters is ideal to avoid unwanted characteristics in recombinant proteins, such as inclusion bodies.12 It has been shown that regulated promoters yield higher quantities of soluble proteins 13. For the BioBrick for AtPFN1, the T7 promoter was chosen because it can be positively regulated through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). DNA sequences downstream of a T7 promoter are transcribed by a T7 RNA polymerase whose expression is induced by the addition of IPTG when working with certain chassis such as E. coli BL21 (DE3).14

LacI regulated Promoter

The T7 promoter is a strong promoter ideal for protein expression induction; nevertheless, excessive production can cause mis-folding and the formation of aggregation bodies.15 Since proper folding of our disulfide-bonded peptides was an important aspect of our genetic design, we opted to use a LacI regulated promoter from the Parts Registry, a weaker yet effective promoter and positively induced by IPTG, for the expression of PsDef1 and WAMP1b.

More Features

6x His-Tag

The addition of a 6x histidine tag to our peptides has the purpose of allowing their purification by immobilized metal affinity purification (IMAC). A 6x His-Tag consist of six histidine residues at the N or C terminus of a recombinant protein. Histidine has an affinity for metal ions, for example Ni2+. IMAC columns allow the isolation of histidine tagged proteins from other compounds in a cell lysate thanks to the metal ions immobilized in a resin inside the columns.16

Erv1p

Erv1p is a protein from Saccharomyces cerevisiae that shows a flavin-linked sulfhydryl oxidase enzymatic activity. This means that it’s capable of oxidizing thiol groups in proteins and catalyzing disulfide bond formation.17 It has been shown that, thanks to its ability to form disulfide bonds de novo. Co-expression of Erv1p allows the proper formation of disulfide bonded proteins in the cytoplasm of E. coli .18 Besides the BioBricks for PsDef1 and WAMP1b, an additional two BioBricks were designed to co-express them along with Erv1p.

Visit our Parts page for more information.

From the Lab to the Field

In order to turn our peptides into a product that can be applied on cotton crops, the following steps will be carried out:

  • Transformation of E. coli chassis with our expression vectors
  • IPTG protein induction
  • Soluble protein extraction
  • Purification by immobilized metal affinity chromatography
  • Nanoencapsulation
  • Packaging
  • Delivery through the irrigation system

Nanoencapsulation

Conventional agrochemical application methods usually result in an intended product overdose, with the purpose of ensuring that the active ingredient (AI) is delivered in sufficient quantities where it is needed.

Unfortunately, more than 90% of the pesticides applied are lost in the environment or are unable to reach the target organism, which causes an increase in cost and more importantly in environmental pollution. Nanotechnology, a science focused on the development of materials on a scale of 1 nm to 100 nm, allows the nanoencapsulation of pesticides and fertilizers for a more effective and efficient AI release.19

Nanoencapsulation consists of the packaging of solid, liquid or gaseous substances in capsules composed of various materials that confer specific properties, such as: efficient delivery, controlled release, greater protection against UV rays, greater solubility and stability, among many others.20

Our nanocapsule


One of the most common materials for nanoencapsulation is the chitosan (CS) copolymer, composed of glucosamine and N-acetylglucosamine. Among its main characteristics are high biocompatibility, biodegradability, low immunogenicity and several biological activities. Given its properties, its use is feasible in the field of agriculture, food and medical industry. On the other hand, polylactic acid (PLA) has been another copolymer widely used in drug delivery systems, since it is of a biodegradable nature and has low hydrophilicity.21

Recently a nanocapsule has been developed from the chitosan-polylactice (CPLA) copolymers, leading to an improvement in the physicochemical properties and biodegradability of the components. Similarly, by controlling the relationship between both polymers, it is possible to obtain a hydrophobic-hydrophilic balance.22

Moreover, the basic character of chitosan is capable of neutralizing acid degradation by polylactic acid products. Also, through several studies, the use of CS and PLA nanocapsules has been tested for encapsulation of both proteins and peptides.23, 24

As for the release of the active ingredients, the CPLA nanocapsule is characterized by being easily degraded in an alkaline environment, indicating a pH-regulated release. Experiments have shown a release rate at a pH of 8.54, of 75% over a period of 10 hours.25 Alluvial soils of cotton crops, have a pH from 6.5 to 9.29 This means that, for WAMP 1b, PsDef and AtPFN1 peptides, the nanocapsule provides a gradual release.

Therefore, based on the previous information, the delivery of V-TION is proposed through a CPLA nanocapsule, providing a pH-regulated release and protection against photodegradation.

General characteristics


Since its components are both hydrophobic and hydrophilic, the resulting nanocapsule is considered amphiphilic. This property promotes the formation of micellar structures, and provides a stabilizing interface between the nucleus of the nanoparticle and the aqueous environment. Also, Nano-mycelia work better than surfactants. The above is especially helpful because a hydrophobic protection will be able to prevent the dispersion of our peptides when applied by irrigation.25

Photodegradation


In addition to this, it is important to consider that the soil on which cotton is developed has optimal temperatures of 27°C - 32°C. However, temperatures of up to 44°C have been reported.26 Individually, chitosan has a melting point of 88°C, while polylactic acid withstands temperatures up to 160°C. Overall, the nanocapsule made from both materials has been exposed to irradiation using a 16 W ultraviolet lamp for 150 min, verifying that its usage largely prevents the degradation of the active compound.25, 27, 28

Biodegradability and toxicity


Finally, you cannot overlook the environmental impact. Taking this into account, another of the great advantages of nanoencapsulation with chitosan and polylactic acid is its high biodegradability and virtually nonexistent toxicity.

There have been identified organisms, from prokaryotes to animals, plants and yeasts, that have a wide variety of enzymes capable of degrading chitosan. When this degradation occurs, amino-sugars or non-toxic compounds are released. Likewise, CS is also considered a biocompatible compound, that means that when in contact with a living system it has no adverse effects. An LD50 toxicity of 16 g/kg has been reported, which is similar to the toxicity of salt and sugar; this is considered minimal toxicity and is safe for both plants and animals.21

As for polylactic acid, it is biodegradable, meaning it degrades naturally when exposed to the environment. In addition to its low toxicity, it has been approved by the US Food and Drug Administration.28, 30

Inert Ingredients Formulation

Nanoencapsulation is followed by a lyophilization process that allows the presentation of the product as a wettable powder (WP). The WP formulation is a powder capable of forming a suspension while being mixed with water before its application. WP formulations are composed by one or more active ingredients that are combined with inert substances, diluents and surfactants. In this case, wetting agents are used to help with the suspension of water particles. A dispersing agent is also added to avoid its flocculation before its application.31

First, the right humectant is needed for the production of the powder. In this case, the agent helps with the dilution process when the powder is applied to water, reducing superficial tension which makes it possible for the lyophilized powder to easily mix with water, as a wetting agent.31 Regarding other ingredients we propose the use of dioctyl sulfosuccinate sodium, which is an commonly used additive as an expressing agent, stabilizer, emulsifiable and moisturizer; and sodium N-methyl oleoyl taurate which is an anionic surfactant.32,33 A dispersant is also needed in the composition of the wettable powder. In this case, the dispersant’s function is to maintain a stable suspension and avoid particle agglomeration when two components are mixed, we plan on using a modified styrene acrylic polymer which is a polymer dispersant in powder form specially recommended for WP.31

The use of a surfactant is also needed, which is a key component in the composition of the wettable powder. In this case, the main focus are its dispersion and humidification properties. We propose the use of an anionic surfactant, this will be used thanks to its amphiphilic properties since molecules with amphiphilic character have the ability to soluble polar and non-polar molecules.34 The use of biodegradable surfactants like alkylphenol ethoxylate, alcohol ethoxylates and high oil fatty acids limits the formation of foam during the mixing process.31

In order to define the percentage of our inert and our active ingredients that are shown in our label, we took as reference a product in the market that was similar to our production in its composition, which is “Harpin EA”. This product also has a soluble powder formulation, which contains a type of protein that is naturally produced in some bacteria and are used to improve the health of plants, production, and quality of the crops.35 Due to the fact that this product was used as our reference, we decided to propose the following percentage: 3% of the active ingredient and 97% of inert ingredient, this percentage is commonly used in the vast majority of products with a hairpin protein as an active ingredient. 36,37

Application

For our products application, we took the client’s needs as our main focus, this led to the most effective application method which is through an irrigation system. Therefore, high degrees of automatization can be achieved when looking at labor, water and energy savings. Another advantage is that it does not need leveling since it is capable to adapt to undulation topography, which allows a better planification during sowing season. This is called an agriculture insurance since it does not require rain to its growth.38 It also allows the application of products directly on the plant’s base near the roots for better exploitation in less time, which is an advantage for our product. It enables a rational and measured use of water in agricultural fields, avoiding wet roads and areas that surround the crops, reducing weed growth and disease development. Finally, it multiplies water and chemical product exploitation applying them systematically and directly.39 Our product dissolves in the preparation tank to be injected by the use of injection pumps into the irrigation system. For more information go to our Entrepreneurship page.

Spore removal before sowing is possible by having antifungal peptides that posses different action mechanisms. PsDef1 and WAMP1b are capable of attacking the fungus when the plant is already infected, and AtPFN1 allows spore removal even before sowing. A biofungicide composed of the three peptides would be able to act as a preventive and treating product, likewise, when acting inside the infected organism it can be classified as a systemic biofungicide. Because of this, the use of our product in two applications is proposed, the first is preventive and used during the initial growth process or seedling phase that lasts 12-20 days after sowing, this is the cycle stage where the root starts to grow and our product can start bringing protection to the plant.40 The second application would be performed at least two weeks after flowering, 30-40 days after sowing 40, because it has been proven that products used in cotton have higher effectivity in this stage; after flowering disease treatment becomes complicated.40 Besides, V. dahliae symptoms are frequently seen in plants that are in the flowering stage and even more developed ones. 41

Our product will be applied by medium watering with an average watering of 6 hours, therefore, our product would be applied once the first 3 watering hours are completed. This would be done aiming at the roots, covering them completely with our peptides. The injection of our product will stop half an hour before watering ends, with the purpose of not losing product on the surface. In conclusion, our product should be applied for 2 and a half-hour period of irrigation. This would result in an amount of 25 m3 of water per hectare per hour, which is the average water invested in a cotton field with a common drip tape irrigation system.42

Packaging

Thanks to the classification of the product, it turned out to be necessary to explain the sanitary conditions of the containers and package. Analyzing the previous factors, it was concluded that the ideal container is a “sachet” which is commonly used for powdered pesticides. Because it is hermetic, it guarantees the conservation and purity of the product. Thanks to the aluminium, paper, and other plastic components of the package, the bag possesses flexibility combined with antimicrobial properties with high levels of asepsia in order to extend the conservation of the product.43 Due to the fact that this product is widely used in different species including annual plants, herbaceous perennials and wood, usage of the product will probably imply large amounts of it.44 The following NOM was taken into account: NOM-018-STPS-2015 (NOM for the identification and communication of dangers and risks by dangerous chemical substances in work centers) in order to define the conditions where the product can be stored while being unused.45 For more information visit our Risk Analysis.

References

  1. Keymanesh, K., Soltani, S., & Sardari, S. (2009). Application of antimicrobial peptides in agriculture and food industry. World Journal of Microbiology and Biotechnology, 25(6), 933-944. doi: 10.1007/s11274-009-9984-7
  2. Berlanger, I., & Powelson, M. (2005). Verticillium wilt. The Plant Health Instructor. doi: 10.1094/PHI-I-2000-0801-01
  3. Park, S.-C., Kim, I. R., Kim, J.-Y., Lee, Y., Kim, E.-J., Jung, J. H., … Lee, J. R. (2018). Molecular mechanism of Arabidopsis thaliana profilins as antifungal proteins. Biochimica et Biophysica Acta (BBA) - General Subjects, 1862(12), 2545–2554. doi:10.1016/j.bbagen.2018.07.028
  4. Fradin, E., & Thomma, B. (2006). Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V.albo-atrum. Molecular Plant Pathology, 7(2), 71-86. doi: 10.1111/j.1364-3703.2006.00323.x
  5. Khairutdinov, B. I., Ermakova, E. A., Yusypovych, Y. M., Bessolicina, E. K., Tarasova, N. B., Toporkova, Y. Y., … Nesmelova, I. V. (2017). NMR structure, conformational dynamics, and biological activity of Ps Def1 defensin from Pinus sylvestris. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1865(8), 1085–1094. doi:10.1016/j.bbapap.2017.05.012
  6. Naumann, T. A., & Wicklow, D. T. (2013). Chitinase modifying proteins from phylogenetically distinct lineages of Brassica pathogens. Physiological and molecular plant pathology, 82, 1-9. doi: https://doi.org/10.1016/j.pmpp.2012.12.001
  7. Slavokhotova, A. A., Naumann, T. A., Price, N. P., Rogozhin, E. A., Andreev, Y. A., Vassilevski, A. A., & Odintsova, T. I. (2014). Novel mode of action of plant defense peptides–hevein‐like antimicrobial peptides from wheat inhibit fungal metalloproteases. The FEBS journal, 281(20), 4754-4764. doi: 10.1111/febs.13015
  8. Veggiani, G., & de Marco, A. (2011). Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase. Protein expression and purification, 79(1), 111-114. doi: 10.1016/j.pep.2011.03.005
  9. New England Biolabs. (2017). FAQ: What is the difference between BL21 and BL21(DE3) competent E.coli cells? Recovered from: https://international.neb.com/faqs/2016/01/21/what-is-the-difference-between-bl21-and-bl21-de3-competent-e-coli-cells2
  10. New England Biolabs. (2017). BL21 (DE3) Competent E. coli. New England Biolabs. Recovered from: https://international.neb.com/-/media/catalog/datacards-or-manuals/c2530datasheet-lot5.pdf?rev=b24342f0b57e4efc82499efee0a57aa3
  11. Berkmen, M. (2012). Production of disulfide-bonded proteins in Escherichia coli. Protein expression and purification , 82(1), 240-251. doi: https://doi.org/10.1016/j.pep.2011.10.009
  12. Banerjee, A., Leang, C., Ueki, T., Nevin, K. P., & Lovley, D. R. (2014). Lactose-inducible system for metabolic engineering of Clostridium ljungdahlii. Applied and environmental microbiology, 80(8), 2410–2416. doi:10.1128/AEM.03666-13
  13. Terpe, K. (2006). Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Applied microbiology and biotechnology, 72(2), 211. doi: 10.1007/s00253-006-0465-8
  14. New England BioLabs. (2012). E. coli Expression Strains. Retrieved from https://international.neb.com/products/competent-cells/e-coli-expression-strains/e-coli-expression-strains
  15. Ren, G., Ke, N., & Berkmen, M. (2016). Use of the SHuffle strains in production of proteins. Current protocols in protein science , 85(1), 5-26.
  16. Bio-Rad. (2019). His-Tag Purification. Retrieved from https://www.bio-rad.com/featured/en/his-tag-purification.html
  17. Lee, J. E., Hofhaus, G., & Lisowsky, T. (2000). Erv1p from Saccharomyces cerevisiae is a FAD‐linked sulfhydryl oxidase. FEBS letters, 477(1-2), 62-66. doi: 10.1016/s0014-5793(00)01767-1
  18. Hatahet, F., Nguyen, V. D., Salo, K. E., & Ruddock, L. W. (2010). Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microbial cell factories, 9(1), 67. doi: 10.1186/1475-2859-9-67
  19. Kah, M., & Hofmann, T. (2014). Nanopesticide research: Current trends and future priorities. Environment International, 63, 224–235. doi:10.1016/j.envint.2013.11.015
  20. Ali MA, Rehman I, Iqbal A, Din S, Rao AQ, Latif A, Samiullah TR, Azam S, Husnain T. (2014). Nanotechnology, a new frontier in Agriculture. Adv. life sci., 1(3), pp. 129-138.
  21. Lizardi-Mendoza, J., Argüelles Monal, W. M., & Goycoolea Valencia, F. M. (2016). Chemical Characteristics and Functional Properties of Chitosan. Chitosan in the Preservation of Agricultural Commodities, 3–31. doi:10.1016/b978-0-12-802735-6.00001-x
  22. Jie, P., Venkatraman, S. S., Min, F., Freddy, B. Y. C., & Huat, G. L. (2005). Micelle-like nanoparticles of star-branched PEO–PLA copolymers as chemotherapeutic carrier. Journal of Controlled Release, 110(1), 20–33. doi:10.1016/j.jconrel.2005.09.011
  23. Prego, C., Torres, D., & Alonso, M. J. (2005). The potential of chitosan for the oral administration of peptides. Expert Opinion on Drug Delivery, 2(5), 843–854. doi:10.1517/17425247.2.5.843
  24. Chandy, T., Das, G. S., Wilson, R. F., & Rao, G. H. R. (2002). Development of polylactide microspheres for protein encapsulation and delivery. Journal of Applied Polymer Science, 86(5), 1285–1295. doi:10.1002/app.11139
  25. Xu, L., Cao, L.-D., Li, F.-M., Wang, X.-J., & Huang, Q.-L. (2014). Utilization of Chitosan-Lactide Copolymer Nanoparticles as Controlled Release Pesticide Carrier for Pyraclostrobin AgainstColletotrichum gossypiiSouthw. Journal of Dispersion Science and Technology, 35(4), 544–550. doi:10.1080/01932691.2013.800455
  26. Saucedo, A. (2018). Registra Ojinaga la más alta temperatura. Retrieved from: https://www.elheraldodechihuahua.com.mx/local/registra-ojinaga-la-mas-alta-temperatura-1865744.html
  27. LookChem. 2010. Chitosan supplier. Retrieved from: https://www.lookchem.com/Chitosan/
  28. Rogers, T. (2015). Everything you need to know about polylactic acid (PLA). Retrieved from: https://www.creativemechanisms.com/blog/learn-about-polylactic-acid-pla-prototypes
  29. NAANDANJAIN. (2014). Algodón. Retrieved from: http://es.naandanjain.com/uploads/catalogerfiles/Coton/NDJ_Cotton_booklet_span_060314F_72.pdf
  30. Ultimaker. (2017). Ficha de datos de seguridad PLA. Retrieved from: https://ultimaker.com/download/67584/SDS%20PLA%20v3.004-spa-ES.pdf
  31. CRODA. (2019). Productos y Aplicaciones. Polvo mojable. CROPCARE. Retrieved from: https://www.crodacropcare.com/es-mx/products-and-applications/wettable-powder#tab-collapse-dispersant
  32. Badui, S. (2006). Química de los alimentos. México: Pearson Educación.
  33. Brockhaus, ABC., & Chemie, VEB. F. A. (1965). Brockhaus Verlag Leipzig. 503−504.
  34. Sanz, A. (2014). La industria de los agentes tensoactivos. Química orgánica industrial. Retrieved from: https://www.eii.uva.es/organica/qoi/tema-10.php
  35. Terralia. (2009). Proteína Harpin 3%. PS. Información Agrícola. Retrieved from: https://www.terralia.com/agroquimicos_de_mexico/view_composition?composition_id=14026
  36. Plant Health. (2012a). Tecnología Harpin 3%.Plant Health Care. Retrieved from: http://www.planthealthcare.es/tecnologias/harpin/
  37. Plant Health. (2012b). Proteína Harpin ea. Messenger STS. Plant Health Care. Retrieved from: http://www.tacsa.mx/DEAQ/src/productos/1334_132.htm
  38. Campos del mañana. (2011). Ventajas de instalar un sistema de riego en su campo. Retrieved from: https://cmsa.com.py/riego/ventajas-sistema-de-riego/
  39. Sistema Agrícola. (2016). Quimigación: Químicos en el sistema de riego. Retrieved from: http://sistemaagricola.com.mx/blog/quimigacion-en-el-sistema-de-riego/
  40. SAGARPA. (2002). Generalidades del cultivo del algodonero. Gobierno de Baja California. Retrieved from: http://www.oeidrus-bc.gob.mx/sispro/algodonbc/Descargas/algodon.pdf
  41. Bonacic, I., Fogar, M., Guevara, C., Simonella, M. Algodón Manual, de campo. Retrieved from: http://rian.inta.gov.ar/agronomia/Manual_Algodon.pdf
  42. García, C. Personal Communication, September 25, 2019.
  43. QuimiNet. (2012). El empaque tipo sachet. QuimiNet. Retrieved from: https://www.quiminet.com/articulos/el-empaque-tipo-sachet-2675644.htm
  44. Dirección General de Sanidad Vegetal. (2018). Protocolo de Diagnóstico: Verticillium dahliae. Tecámac, Estado de México: SENASICA. Retrieved from: https://www.gob.mx/cms/uploads/attachment/file/391130/4._Protocolo_Verticillium_dahliae.V01.pdf.
  45. Sistema armonizado para la identificación y comunicación de peligros y riesgos por sustancias químicas peligrosas en los centros de trabajo. Norma Oficial Mexicana NOM-018-STPS-2015. Diario Oficial de
    la Federación, October 09, 2015.

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