Quitosano. Características químicas y físicas

El quitosano, es un polisacárido que se obtiene a partir de la quitina, segundo polisacárido más abundante en la naturaleza. Es un copolímero líneal formado por residuos de unidades de D-glucosamina en mayor medida y N-acetil D-glucosamina en menor medida, distribuidos aleatoriamente y unidos por enlaces β 1,4. Según la Unión Internacional de Química Pura y Aplicada (IUPAC), es 2 amino 2 desoxi-D-glucopiranosa (D-glucosamina GlcN) y 2 acetamida- 2 desoxi- D glucopiranosa N-acetil glucosamina (11. Harish Prashanth KV, Tharanathan RN. Chitin/chitosan: modifications and their unlimited application potential-an overview. Trends in Food Science and Technology. 2007;18(3):117-31. https://doi.org/10.1016/j.tifs.2006.10.022 ). Tanto el contenido como la secuencia de estas unidades determinarán las propiedades físico-químicas y biológicas de este polímero. El quitosano tiene un contenido de nitrógeno (N) mayor que 7 y posee una distribución regular de los grupos aminos libres, que pueden ser protonados por ciertos ácidos, cargándose positivamente y esto le da un carácter policatiónico. Este hecho permite explicar algunas de sus propiedades como son la habilidad de enlazarse con sustancias cargadas negativamente, tales como lípidos, proteínas, colorantes entre otras; así como floculante, adherente y adsorbente, adicionales a las reacciones típicas de las aminas (99. Muzzarelli RAA. Enzymatic synthesis of chitin and chitosan. Occurrence of chitin. In: Muzzarelli RAA, editor. Chitin [Internet]. Pergamon; 1977 [cited 28/08/2021]. p. 5-44. https://doi.org/10.1016/B978-0-08-020367-6.50007-6 ). Al unirse los grupos amino del quitosano a los aminoácidos ácidos de las proteínas se produce las interacciones electrostáticas que conlleva a desórdenes celulares en los microorganismos.

Este biopolímero posee actividad antimicrobiana contra una amplia variedad de microrganismos incluyendo hongos, algas y algunas bacterias. Su funcionalidad y actividad depende de sus características como: masa molecular, grado de acetilación, célula huésped, presencia de nutrientes naturales, composición química o nutricional de los sustratos y condiciones ambientales. En este sentido, respecto al grado de acetilación se puede plantear que mientras menor sea el grado de acetilación, mayor es la actividad antimicrobiana (1010. Kong M, Chen X, Xue Y, Liu C, Yu L, Ji Q, et al. Preparation and antibacterial activity of chitosan microshperes in a solid dispersing system. Frontiers of Materials Science in China [Internet]. 2008;2(2):214-20. Available from: https://link.springer.com/content/pdf/10.1007/s11706-008-0036-2.pdf ,1111. Takahashi T, Imai M, Suzuki I, Sawai J. Growth inhibitory effect on bacteria of chitosan membranes regulated with deacetylation degree. Biochemical Engineering Journal [Internet]. 2008;40(3):485-91. Available from: https://www.sciencedirect.com/science/article/abs/pii/S1369703X08000430 ).

Pyricularia oryzae agente causal del añublo o quemazón del arroz

Este patógeno de plantas es muy eficaz ya que puede reproducirse sexualmente (teleomorfo: Magnaporthe grisea Barr (It. Hebert) sin Magnaporthe oryzae) y asexualmente (anamorfo: Pyricularia oryzae). El Grupo de Trabajo Pyricularia/Magnaporthe ha establecido bajo los auspicios de la Comisión Internacional sobre la Taxonomía de Hongos (CITH) la posibilidad de conservar el nombre Magnaporthe sobre Pyricularia. Sin embargo, dicha conservación requiere un cambio en la especie tipo del género Magnaporthe y causaría numerosos cambios de nombres para esas especies actualmente ubicadas en Pyricularia (1212. Zhang N, Luo J, Rossman AY, Aoki T, Chuma I, Crous PW, et al. Generic names in Magnaporthales. IMA fungus [Internet]. 2016;7(1):155-9. https://doi.org/10.5598/imafungus.2016.07.01.09 ).

El nombre tipificado asexualmente Pyricularia es el nombre correcto para el hongo que produce la quemazón del arroz, que se corresponde bien con la patogenicidad y las características ecológicas y evolutivas. Por lo tanto, el nombre Pyricularia oryzae debe usarse para el hongo que produce esta enfermedad. Sin embargo, el sinónimo Magnaporthe oryzae puede seguir siendo mencionado en publicaciones como Pyricularia oryzae (syn. Magnaporthe oryzae). Esta práctica ayudará a cerrar una brecha potencial en la literatura y el conocimiento de esta importante especie (1212. Zhang N, Luo J, Rossman AY, Aoki T, Chuma I, Crous PW, et al. Generic names in Magnaporthales. IMA fungus [Internet]. 2016;7(1):155-9. https://doi.org/10.5598/imafungus.2016.07.01.09 ). En cuanto a grisea y oryzae, son especies muy distintas. Grisea es para cepas de Digitaria y oryzae para cepas de arroz, trigo y otras gramíneas; aunque en la literatura se consideran sinónimos y se utilizan los cuatros nombres: Pyricularia oryzae, Magnaporthe oryzae, Pyricularia grisea y Magnaporthe grisea.

Pyricularia oryzae conocida como piriculariosis, añublo, quemazón, fallada, mancha, bruzone del arroz o tizón foliar es una de las enfermedades más serias del cultivo de arroz (Oryza sativa L.), la cual ha causado significantes pérdidas en los rendimientos a nivel mundial (1313. Lugo L, Jayaro Y, González Á, Borges O. Identification of sources of partial resistance to Pyricularia grisea in rice cultivars and experimental lines. Fitopatología Venezolana [Internet]. 2008;21(2):51-8. Available from: https://www.cabdirect.org/cabdirect/abstract/20093204367 ).

La distribución del añublo es mundial ya que se encuentra en todos los agroecosistemas de los trópicos y de las zonas templadas en que se cultiva el arroz comercial. La piriculariosis genera grandes pérdidas en la producción del grano, tanto en el sistema de secano como en el de riego. Esta enfermedad se ha reportado en al menos 85 países de todo el mundo. Se descubrió en Italia en 1560 y más tarde fue encontrada en China (1637), Japón (1760), Estados Unidos (1960) y la India (1913) (1414. Rossman AY, Howard RJ, Valent B. Pyricularia grisea the correct name for the rice blast disease fungus. Mycologia [Internet]. 1990;82(4):509-12. Available from: https://www.tandfonline.com/doi/abs/10.1080/00275514.1990.12025916?journalCode=umyc20 ). La piriculariosis ha causado significantes pérdidas, por ejemplo: en la India el 90 % (1515. Manjunatha B, Krishnappa M. Morphological characterization of Pyricularia oryzae causing blast disease in rice (Oryza sativa L.) from different zones of Karnataka. Journal of Pharmacognosy and Phytochemistry [Internet]. 2019;8(3):3749-53. Available from: https://www.phytojournal.com/archives/2019/vol8issue3/PartBC/8-3-279-550.pdf ), en China el 70 % (1616. Ou SH. Rice diseases [Internet]. IRRI; 1985. Available from: https://books.google.es/books?hl=es&lr=&id=-k3mewv9nMoC&oi=fnd&pg=PR1&dq=Rice+Diseases&ots=Zmj_rBBc8i&sig=C413L03O2pB85A2EhfQhPD1KApU#v=onepage&q=Rice%20Diseases&f=false ), en Tailandia fue afectada en 1987 solamente 1900 ha, ya en 1988 se incrementó a 490 000 ha, en España y en la zona del mediterráneo también ha causado daños (1717. Koutroubas SD, Katsantonis D, Ntanos DA, LUPOTTO E. Blast disease influence on agronomic and quality traits of rice varieties under Mediterranean conditions. Turkish Journal of Agriculture and forestry [Internet]. 2009;33(5):487-94. Available from: https://journals.tubitak.gov.tr/agriculture/abstract.htm?id=10484 ). México, por su parte ha reportado un abatimiento de la producción hasta del 30 % en siembras de temporal. Cuba ha sido otro de los países donde ha causado daños este patógeno y cuando las condiciones son favorables se ha incrementado las pérdidas hasta un 70 % (66. Rodríguez AT, Ramírez MA, Cárdenas RM, Hernández AN, Velázquez MG, Bautista S. Induction of defense response of Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating seeds with chitosan and hydrolyzed chitosan. Pesticide Biochemistry and Physiology [Internet]. 2007;89(3):206-15. https://doi.org/10.1016/j.pestbp.2007.06.007 ).

Clasificación taxonómica, morfología y sintomatología de Pyricularia oryzae

El agente causal de la piriculariosis se clasifica taxonómicamente en la clase: Deuteromicetes, orden: Moniliales, familia: Dematiaceae, género: Pyricularia, especie: Pyricularia oryzae. Pyricularia oryzae posee conidióforos simples, tabicados y de color pardusco (Figura 1A). Los conidióforos nacen solitarios o en grupos de tres y en sus extremos llevan los conidios. Los conidióforos nacen solitarios o en grupos de tres y en sus extremos llevan los conidios, los cuales son hialinos, fusiforme y están divididos por dos septos en forma equidistantes.

Es una enfermedad compleja debido a la variabilidad patogénica y a la rapidez con la que este hongo vence la resistencia de la planta de arroz. El micelio del hongo produce una sustancia tóxica conocida como piricularina (1616. Ou SH. Rice diseases [Internet]. IRRI; 1985. Available from: https://books.google.es/books?hl=es&lr=&id=-k3mewv9nMoC&oi=fnd&pg=PR1&dq=Rice+Diseases&ots=Zmj_rBBc8i&sig=C413L03O2pB85A2EhfQhPD1KApU#v=onepage&q=Rice%20Diseases&f=false ), que inhibe el crecimiento de los tejidos y los desorganiza. Ataca las partes aéreas de la planta como las hojas, tallos, nudos y espigas (1818. Kulmitra AK, Sahu N, Sahu MK, Kumar R, Kushram T, Sanath Kumar VB. Growth of Rice blast fungus Pyricularia oryzae (Cav.) on different solid and liquid media. International Journal of Current Microbiology and Applied Sciences [Internet]. 2017;6(6):1154-60. https://doi.org/10.20546/ijcmas.2017.606.133 ) (Figuras 1B,C). El hongo produce unas manchas o lesiones en las hojas de forma alargadas o elípticas a romboides y de color marrón uniforme que más tarde cambiarán a un color grisáceo en la parte central, hecho que indica la esporulación del hongo; aunque su tamaño y color varían de acuerdo con las condiciones ambientales y con la susceptibilidad de las variedades (1919. Cárdenas RM, Pérez N, Cristo E, González MC, Fabré L. Estudio sobre el comportamiento de líneas y variedades de arroz (Oryza sativa Lin.) ante la infección por el hongo Pyricularia grisea Sacc. Cultivos Tropicales [Internet]. 2005;26(4):83-7. Available from: https://www.redalyc.org/pdf/1932/193216160012.pdf ). Su temperatura óptima de crecimiento está entre 22-29 °C y una elevada humedad relativa entorno al 90 %, que coinciden plenamente con las condiciones climáticas de países tropicales. La presencia de elevadas concentraciones de nitrógeno en el agua favorece también el desarrollo del hongo y produce gran cantidad de esporas. Las esporas llegan desde los restos de cosecha de la temporada anterior o de malas hierbas donde ha estado alojado el hongo durante el invierno (2020. Cárdenas RM, Polón CR, Pérez N, Cristo E, Mesa S, Fabré L, et al. Relación entre la incidencia de la piriculariosis (Pyricularia grisea Sacc.) del arroz (Oryza sativa Lin.) y diferentes variables climáticas en el Complejo Agroindustrial Arrocero Los Palacios. Cultivos Tropicales [Internet]. 2010;31(1):00-00. Available from: http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S0258-59362010000100002 ).

Estrategias generales para el control de Pyricularia oryzae

El control de la enfermedad se basa, principalmente, en la aplicación de fungicidas químicos sistémicos entre los que se cuentan los del grupo de los benzimidazoles, como el procloraz, tebuconazol y propiconazol, entre otros. Sin embargo, su uso ha presentado varias desventajas, ya que ocasionan contaminación del manto freático y de los cuerpos de agua colindantes, generando efectos nocivos sobre diversos organismos. Además, es importante señalar, que estos fungicidas químicos están siendo vulnerados por el hongo, a causa del surgimiento de poblaciones con pérdida de sensibilidad a su modo de acción (2121. Manzo Sánchez G, Carrillo Madrigal H, Guzmán González S, Orozco Santos M. Análisis de la Sensibilidad in vitro de Mycosphaerella fijiensis, Agente Causal de la Sigatoka Negra del Banano a los Fungicidas Benomyl, Propiconazol y Azoxistrobin. Revista mexicana de fitopatología [Internet]. 2012 [cited 28/08/2021];30(1):81-5. Available from: http://www.scielo.org.mx/scielo.php?script=sci_abstract&pid=S0185-33092012000100008&lng=es&nrm=iso&tlng=es ). Debido a esta situación se viene presentando un número creciente de ciclos de fungicidas por año para proteger los cultivos contra esta enfermedad, lo cual implica un incremento en los costos de producción y en efectos negativos de los fungicidas convencionales sobre el ambiente, cuestionando su uso comercial, siendo entonces prioritario la búsqueda de alternativas que permitan complementar al manejo integrado de las enfermedades.

Actividad in vitro e in vivo del quitosano y sus derivados sobre Pyricularia oryzae

La propiedad antimicrobiana del quitosano y sus derivados han recibido considerable atención en años recientes, debido al inminente problema asociado con los agentes químicos sintéticos. Estos compuestos han demostrado ser fungicidas y fungistáticos para el control de Botrytis cinerea, Aspergillus flavus, Aspergillus parasiticus, Drechstera sorokiana, Fusarium acuminatum, Fusarium graminearum, Micronectriella nivalis, Rhizoctonia solani, Alternaria alternata, Colletotrichum gloesporioides, Penicillum spp., Fusarium oxysporum y Bipolaris oryzae, entre otros hongos (2222. Hua C, Li Y, Wang X, Kai K, Su M, Zhang D, et al. The effect of low and high molecular weight chitosan on the control of gray mold (Botrytis cinerea) on kiwifruit and host response. Scientia Horticulturae [Internet]. 2019;246:700-9. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0304423818308197 -2626. Rodríguez Pedroso AT, Plascencia Jatomea M, Bautista Baños S, Cortez Rocha MO, Ramírez Arrebato MÁ. Actividad antifúngica in vitro de quitosanos sobre Bipolaris oryzae patógeno del arroz. Acta Agronómica [Internet]. 2016;65(1):98-103. Available from: http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0120-28122016000100014 ).

Al respecto, la actividad antifúngica del quitosano y sus derivados ha sido observada en diferentes estados de desarrollo de los hongos como la afectación al crecimiento y el desarrollo del agente patógeno, esporulación, viabilidad y germinación de las esporas y la producción de factores de virulencias fúngicos (2323. Sánchez-Domínguez D, Ríos MY, Castillo-Ocampo P, Zavala-Padilla G, Ramos-García M, Bautista-Baños S. Cytological and biochemical changes induced by chitosan in the pathosystem Alternaria alternata-tomato. Pesticide biochemistry and physiology [Internet]. 2011;99(3):250-5. Available from: https://d1wqtxts1xzle7.cloudfront.net/44367734/Cytological_and_biochemical_changes_indu20160403-12579-18vumyq.pdf?1459730343=&response-content-disposition=inline%3B+filename%3DCytological_and_biochemical_changes_indu.pdf&Expires=1631769785&Signature=IGGgjxkuWLCyHKJnBKWJkxGt1dhC3jDsalPwe033Zh69KP~oMc9leISiFCA0WSiKUCxKb1K~RCJkP0URuU~09UxsZWRx6yjMBZ-~tCQYVGBeFq4iuV~0LuAXIeh1ysKZ53HVKZb0Z2fVsYsTRC~FMcGsjqZbRwVPD1mjM7VE43h3RvolAkQ4LLzRWs1KSSscUcUHoy-8cVMHx~zmJFU7DCf~ZGlzwERIsr7TgSW2w7xT7vzMf-OaiiFMda~v241ukzc5Vd6HBXZUxr1J16f266xhUCV5w6uDBnm4X3MHMaFFjIaf9XPSJdfnmDnR8TAzi11jPKfVHXjjuNPU0Y8Zg&Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA ,2727. Živković S, Stevanović M, Đurović S, Ristić D, Stošić S. Antifungal activity of chitosan against Alternaria alternata and Colletotrichum gloeosporioides. Pesticidi i fitomedicina [Internet]. 2018;33(3-4):197-204. Available from: https://plantarum.izbis.bg.ac.rs/bitstream/handle/123456789/543/541.pdf?sequence=1 ,2828. Badawy ME, Rabea EI. A biopolymer chitosan and its derivatives as promising antimicrobial agents against plant pathogens and their applications in crop protection. International Journal of Carbohydrate Chemistry [Internet]. 2011;2011. https://doi.org/10.1155/2011/460381 ). Algunos autores han comprobado el efecto fungicida de estos compuestos a diferentes concentraciones y aislamientos de P. grisea (55. Rodríguez AT, Ramírez MA, Nápoles MC, Márquez R, Cárdenas RM. Antifungal activity of chitosan and one of its hydrolysates on Pyricularia grisea, Sacc. Fungus. Cultivos Tropicales [Internet]. 2003;24(2):85-8. Available from: https://www.redalyc.org/pdf/1932/193218174015.pdf ,77. Cárdenas RM, Ramírez MA, Rodríguez AT, González LM. Efecto de los derivados de quitina y su combinación con sulfato de cobre en el comportamiento del crecimiento micelial y esporulación de un aislamiento monospórico del hongo Pyricularia grisea Sacc. Cultivos Tropicales [Internet]. 2004;25(4):89-93. Available from: https://www.redalyc.org/pdf/1932/193225911012.pdf ,2929. Rabea EI, Badawy ME, Rogge TM, Stevens CV, Höfte M, Steurbaut W, et al. Insecticidal and fungicidal activity of new synthesized chitosan derivatives. Pest Management Science [Internet]. 2005;61(10):951-60. https://doi.org/10.1002/ps.1085 ). También los oligómeros de quitosano han demostrado tener mejor efecto inhibitorio sobre este patógeno, logrando la concentración mínima inhibitoria a una concentración mayor de 2000 mg L^-1 (3030. Xu J, Zhao X, Han X, Du Y. Antifungal activity of oligochitosan against Phytophthora capsici and other plant pathogenic fungi in vitro. Pesticide Biochemistry and Physiology. 2007;87(3):220-8.).

Estudios llevados a cabo en el laboratorio de Oligosacarinas del Instituto Nacional de Ciencias Agrícolas (INCA) con P. grisea indican que el quitosano y sus oligómeros en el medio de cultivo a la concentración de 1,000 mg L^-1 y pH 5.6, inhibieron totalmente el crecimiento micelial de este hongo (55. Rodríguez AT, Ramírez MA, Nápoles MC, Márquez R, Cárdenas RM. Antifungal activity of chitosan and one of its hydrolysates on Pyricularia grisea, Sacc. Fungus. Cultivos Tropicales [Internet]. 2003;24(2):85-8. Available from: https://www.redalyc.org/pdf/1932/193218174015.pdf ). Sin embargo, es importante considerar el pH de la disolución resultante, que afecta la carga positiva de los grupos aminos, pues en otro ensayo a pH 6 solo hubo una ligera afectación del crecimiento del hongo, aunque se mantuvo una inhibición total de la esporulación (77. Cárdenas RM, Ramírez MA, Rodríguez AT, González LM. Efecto de los derivados de quitina y su combinación con sulfato de cobre en el comportamiento del crecimiento micelial y esporulación de un aislamiento monospórico del hongo Pyricularia grisea Sacc. Cultivos Tropicales [Internet]. 2004;25(4):89-93. Available from: https://www.redalyc.org/pdf/1932/193225911012.pdf ).

Algunos grupos de investigación han comenzado a modificar la molécula del quitosano con adición de grupos hidrofóbicos para aumentar su actividad biológica contra este patógeno. Por ejemplo, N-sulfonada N-sulfobenzoil quitosano (3131. Chen C-S, Liau W-Y, Tsai G-J. Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation. Journal of Food Protection [Internet]. 1998;61(9):1124-8. https://doi.org/10.4315/0362-028X-61.9.1124 ), N,N,N.trimetil quitosano (3232. Jia Z, Xu W. Synthesis and antibacterial activities of quaternary ammonium salt of chitosan. Carbohydrate research [Internet]. 2001;333(1):1-6. https://doi.org/10.1016/S0008-6215(01)00112-4 ) N,O-acyl quitosano (3333. Sashiwa H, Kawasaki N, Nakayama A, Muraki E, Yamamoto N, Zhu H, et al. Chemical modification of chitosan. Synthesis of organosoluble, palladium adsorbable, and biodegradable chitosan derivatives toward the chemical plating on plastics. Biomacromolecules [Internet]. 2002;3(5):1120-5. https://doi.org/10.1021/bm0200478 ), O-acil quitosano (3434. Badawy ME, Rabea EI, Rogge TM, Stevens CV, Steurbaut W, Höfte M, et al. Fungicidal and insecticidal activity of O-acyl chitosan derivatives. Polymer bulletin. 2005;54(4):279-89. https://doi.org/10.1007/s00289-005-0396-z ,3535. Badawy M, Rabea E, Steurbaut W, Rogge T, Stevens C, Smagghe G, et al. Fungicidal activity of some O-acyl chitosan derivatives against grey mould Botrytis cinerea and rice leaf blast Pyricularia grisea. Communications in agricultural and applied biological sciences [Internet]. 2005;70(3):215-8. Available from: https://europepmc.org/article/med/16637180 ), hidroxietil aril quitosano (3636. Ma G, Yang D, Tan H, Wu Q, Nie J. Preparation and characterization of N‐alkylated chitosan derivatives. Journal of applied polymer science [Internet]. 2008;109(2):1093-8. https://doi.org/10.1002/app.28223 ), dimetilpiperazina quitosano (3737. Másson M, Holappa J, Hjálmarsdóttir M, Rúnarsson ÖV, Nevalainen T, Järvinen T. Antimicrobial activity of piperazine derivatives of chitosan. Carbohydrate polymers [Internet]. 2008;74(3):566-71. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0144861708001720 ), carboximetil quitosano (3838. Seyfarth F, Schliemann S, Elsner P, Hipler U-C. Antifungal effect of high-and low- molecular-weight chitosan hydrochloride, carboxymethyl chitosan, chitosan oligosaccharide and N- acetyl-D-glucosamine against Candida albicans, Candida krusei and Candida glabrata. International Journal of Pharmaceutics [Internet]. 2008;353(1-2):139-48. Available from: https://pubmed.ncbi.nlm.nih.gov/18164151/ ), acil urea tiourea quitosano (3939. Zhong Z, Xing R, Liu S, Wang L, Cai S, Li P. Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro. Carbohydrate Research [Internet]. 2008;343(3):566-70. Available from: https://www.researchgate.net/profile/Shengbao-Cai/publication/5765859_Synthesis_of_acyl_thiourea_derivatives_of_chitosan_and_their_antimicrobial_activities_in_vitro/links/5fd7458745851553a0b591d5/Synthesis-of-acyl-thiourea-derivatives-of-chitosan-and-their-antimicrobial-activities-in-vitro.pdf ), N-succinoil quitosano (4040. Tikhonov VE, Stepnova EA, Babak VG, Yamskov IA, Palma-Guerrero J, Jansson H-B, et al. Bactericidal and antifungal activities of a low molecular weight chitosan and its N-/2 (3)-(dodec- 2-enyl) succinoyl/-derivatives. Carbohydrate polymers. 2006;64(1):66-72.) y N-heterocíclico quitosano (4141. Stössel P, Leuba JL. Effect of chitosan, chitin and some aminosugars on growth of various soilborne phytopathogenic fungi. Journal of Phytopathology [Internet]. 1984;111(1):82-90. Available from: https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1439-0434.1984.tb04244.x ). Investigadores han notado que la N-alquilación o N-arilación del quitosano con aldehídos aromáticos o alifáticos aumentaron efectivamente su actividad antifúngica sobre P. grisea (4242. Badawy ME, Rabea EI. Characterization and antimicrobial activity of water-soluble N-(4- carboxybutyroyl) chitosans against some plant pathogenic bacteria and fungi. Carbohydrate polymers [Internet]. 2012;87(1):250-6. Available from: http://damanhour.edu.eg/pdf/researches/1-s2.0-S014486171100645X-main.pdf ).

Con los mismos métodos y técnicas de obtención, pero utilizando diferentes tipos de aldehídos, otros científicos (3131. Chen C-S, Liau W-Y, Tsai G-J. Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation. Journal of Food Protection [Internet]. 1998;61(9):1124-8. https://doi.org/10.4315/0362-028X-61.9.1124 ), observaron actividad antifúngica de 24 nuevos derivados de quitosano (derivados N-benzil quitosanos), los cuales tuvieron mayor efecto inhibitorio que los quitosanos nativos sobre el crecimiento y la formación de esporas de P. grisea, siendo N-(m-nitrobencil) quitosano quien logró el mayor efecto a la concentración de 5 g L^-1. También estos autores observaron que el derivado más activo fue N-(2,2 difeniletil) quitosano, con una concentración mínima inhibitoria de 0,3 g L^-1 contra este patógeno. Por otra parte, grandes avances del quitosano y sus oligómeros sobre el control directo de enfermedades del arroz ha sido observada. Tanto la quitina como el quitosano han demostrado que inducen la acumulación producción de fitoalexinas en este caso de momilactonas A y momilactonas B ante una infección con P. grisea en hojas de arroz a la concentración de 10 µg mL^-1 (4343. Shimizu T, Jikumaru Y, Okada A, Okada K, Koga J, Umemura K, et al. Effects of a bile acid elicitor, cholic acid, on the biosynthesis of diterpenoid phytoalexins in suspension-cultured rice cells. Phytochemistry [Internet]. 2008;69(4):973-81. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0031942207006097?via%3Dihub ).

Un grupo de investigadores publicaron el efecto del quitosano en la estimulación de respuestas de defensa en hojas de arroz (4444. Agrawal GK, Rakwal R, Tamogami S, Yonekura M, Kubo A, Saji H. Chitosan activates defense/stress response (s) in the leaves of Oryza sativa seedlings. Plant Physiology and Biochemistry. 2002;40(12):1061-9.). Después del tratamiento con quitosano al 0,1 %, se observó claramente necrosis en la parte superior de la hoja de arroz. Sin embargo, al tratar plántulas de arroz con 5 mg L^-1 e inocularlas con Magnaporthe grisea 97-23-2D1 se demostró un mejor efecto y control de la enfermedad en más de un 50 % (4545. Lin W, Hu X, Zhang W, Rogers WJ, Cai W. Hydrogen peroxide mediates defence responses induced by chitosans of different molecular weights in rice. Journal of Plant Physiology [Internet]. 2005;162(8):937-44. Available from: http://sippe.ac.cn/gh/papers/caiweiming/Lin2005.pdf ). Sin embargo, en el año 2007 se evaluó, en condiciones semicontroladas, donde trataron semillas de arroz a diferentes concentraciones con dos quitosanos de diferente peso molecular (66. Rodríguez AT, Ramírez MA, Cárdenas RM, Hernández AN, Velázquez MG, Bautista S. Induction of defense response of Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating seeds with chitosan and hydrolyzed chitosan. Pesticide Biochemistry and Physiology [Internet]. 2007;89(3):206-15. https://doi.org/10.1016/j.pestbp.2007.06.007 ). A los 18 días de germinada la semilla las plantas obtenidas fueron inoculadas con esporas de P. grisea, se determinó la actividad de enzimas relacionadas con la defensa como PAL, glucanasa, quitinasa y quitosanasa, observándose un incremento en la actividad de las plantas tratadas con los elicitores, en relación con el control. Además, de no observarse síntomas de la enfermedad en la concentración más elevada utilizada de ambos compuestos (66. Rodríguez AT, Ramírez MA, Cárdenas RM, Hernández AN, Velázquez MG, Bautista S. Induction of defense response of Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating seeds with chitosan and hydrolyzed chitosan. Pesticide Biochemistry and Physiology [Internet]. 2007;89(3):206-15. https://doi.org/10.1016/j.pestbp.2007.06.007 ).

Actualmente, se investiga la aplicación de nanopartículas a base de quitosano con actividad antifúngica y para el control de enfermedades como la piriculariosis (88. Manikandan A, Sathiyabama M. Preparation of chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea. International journal of biological macromolecules [Internet]. 2016;84:58-61. https://doi.org/10. 1016/j.ijbiomac.2015.11.083 ,4646. Pham DC, Nguyen TH, Ngoc UTP, Le NTT, Tran TV, Nguyen DH. Preparation, characterization and antifungal properties of chitosan-silver nanoparticles synergize fungicide against Pyricularia oryzae. Journal of nanoscience and nanotechnology [Internet]. 2018;18(8):5299-305. Available from: https://www.researchgate.net/profile/Dinh-Chuong-Pham/publication/321308729_Preparation_Characterization_and_Antifungal_Properties_of_Chitosan-Silver_Nanoparticles_Synergize_Fungicide-Against-Pyricularia-oryzae/links/5a35486245851532e82f1da0/Preparation-Characterization-and-Antifungal-Properties-of-Chitosan-Silver-Nanoparticles-Synergize-Fungicide-Against-Pyricularia-oryzae.pdf ,4747. Nguyen TH, Thi TV, Nguyen T-T, Le TD, Vo DMH, Nguyen DH, et al. Investigation of chitosan nanoparticles loaded with protocatechuic acid (PCA) for the resistance of Pyricularia oryzae fungus against rice blast. Polymers. 2019;11(1):177.). Por su parte, estudiosos del tema encontraron que nanopartículas de quitosano y plata (Ag) tuvieron una elevada actividad antifúngica sobre Pyricularia oryzae a una concentración de Ag (2 ppm) y de quitosano (4000 ppm) (4646. Pham DC, Nguyen TH, Ngoc UTP, Le NTT, Tran TV, Nguyen DH. Preparation, characterization and antifungal properties of chitosan-silver nanoparticles synergize fungicide against Pyricularia oryzae. Journal of nanoscience and nanotechnology [Internet]. 2018;18(8):5299-305. Available from: https://www.researchgate.net/profile/Dinh-Chuong-Pham/publication/321308729_Preparation_Characterization_and_Antifungal_Properties_of_Chitosan-Silver_Nanoparticles_Synergize_Fungicide-Against-Pyricularia-oryzae/links/5a35486245851532e82f1da0/Preparation-Characterization-and-Antifungal-Properties-of-Chitosan-Silver-Nanoparticles-Synergize-Fungicide-Against-Pyricularia-oryzae.pdf ). Sin embargo, otros científicos observaron que aplicando 500 μl de una solución de nanopartículas de quitosano al 0,1 % sobre hojas de arroz y 24 h más tarde inoculó una suspensión de esporas (1x10⁵ esporas mL^-1) de P. grisea (88. Manikandan A, Sathiyabama M. Preparation of chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea. International journal of biological macromolecules [Internet]. 2016;84:58-61. https://doi.org/10. 1016/j.ijbiomac.2015.11.083 ). A los 10 días no se observaron síntomas de la enfermedad, esto pudo deberse por la estimulación de algún mecanismo de defensa en las hojas y controló la infección (88. Manikandan A, Sathiyabama M. Preparation of chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea. International journal of biological macromolecules [Internet]. 2016;84:58-61. https://doi.org/10. 1016/j.ijbiomac.2015.11.083 ).

Mecanismos de acción del quitosano

Los mecanismos de acción del quitosano no han sido del todo establecidos, aunque existen algunas hipótesis al respecto. En general, las diversas propuestas para explicar la actividad antimicrobiana del quitosano consideran como característica fundamental la naturaleza policatiónica de la molécula, la cual está dada por los grupos NH3+ de la glucosamina, que le confiere importantes propiedades biológicas y fisiológicas (4848. Je J-Y, Kim S-K. Antimicrobial action of novel chitin derivative. Biochimica et Biophysica Acta (BBA)-General Subjects [Internet]. 2006;1760(1):104-9. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0304416505003028?via%3Dihub ,4949. El Hadrami A, Adam LR, El Hadrami I, Daayf F. Chitosan in plant protection. Marine drugs. 2010;8(4):968-87.). En condiciones de pH, el quitosano se comporta como polielectrolito lineal, con un pK alrededor de 6.5, por lo tanto a pH bajos los residuos de glucosamina están cargados positivamente, debido a la protonación de sus residuos aminos, conteniendo una alta densidad de cargas positivas, lo que le permite unirse fuertemente a superficies cargadas negativamente (5050. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM. Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryotic Cell [Internet]. 2005;4(4):703-15. Available from: https://journals.asm.org/doi/pdf/10.1128/EC.4.4.703-715.2005 ). Se plantea que cuando la carga positiva sobre el C-2 del monómero de glucosamina se encuentra por debajo de pH 6, del quitosano es más soluble y tiene una mejor actividad antimicrobiana que la quitina (3131. Chen C-S, Liau W-Y, Tsai G-J. Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation. Journal of Food Protection [Internet]. 1998;61(9):1124-8. https://doi.org/10.4315/0362-028X-61.9.1124 ,5151. Velásquez CL. Some potentialities of chitin and chitosan for uses related to agriculture in Latin America. Revista Científica UDO Agrícola. 2008;8(1):1-22.).

Otro mecanismo propuesto es la interacción entre la carga positiva de la molécula de quitosano y la carga negativa de las células de la membrana microbiana que conduce a la salida de proteínas y otros constituyentes intracelulares (3131. Chen C-S, Liau W-Y, Tsai G-J. Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation. Journal of Food Protection [Internet]. 1998;61(9):1124-8. https://doi.org/10.4315/0362-028X-61.9.1124 ). El más abundante de los esfingolípidos es el manosildiinositolfosfato-ceramida (M(IP2)C), el cual presenta dos cargas negativas. Estas cargas negativas correspondientes a la membrana plasmática (M(IP2)C, pueden unirse a los grupos aminos de los residuos de glucosamina del quitosano. En otros estudios se ha observado, que el quitosano forma canales de transporte de moléculas en bicapas lipídicas artificiales, lo que provee evidencia de que este compuesto puede desorganizar a la membrana celular (5050. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM. Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryotic Cell [Internet]. 2005;4(4):703-15. Available from: https://journals.asm.org/doi/pdf/10.1128/EC.4.4.703-715.2005 ). Por su parte, un grupo de trabajo analizaron el modo de acción del quitosano sobre las células fúngicas y observaron dos aspectos: que el quitosano permeabiliza la membrana plasmática del hongo y penetra a las células del hongo, proceso que es dependiente de ATP y también demostró que diferentes tipos de células (conidio, tubo germinativo e hifa) exhiben diferente sensibilidad al quitosano (5252. Palma-Guerrero J, Huang I-C, Jansson H-B, Salinas J, Lopez-Llorca LV, Read ND. Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner. Fungal Genetics and Biology [Internet]. 2009;46(8):585-94. Available from: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.458.5391&rep=rep1&type=pdf ,5353. López Jiménez JÁ, Palma Guerrero J, Pérez Berná AJ, Huang IC, Jansson HB, Salinas J, et al. Membrane fluidity determines sensitivity of filamentous fungi to chitosan. Molecular Microbiology, n^o75, vol. 4 [Internet]. 2010; Available from: https://digitum.um.es/digitum/bitstream/10201/38147/1/Membrane%20fluidity%20determines%20sensitivity.pdf ). En el año 2010, el mismo grupo de investigadores demostró, a través de técnicas biológicas, bioquímicas, genéticas y biofísicas, que la actividad antifúngica del quitosano depende de la fluidez de la membrana plasmática del hongo, la cual está determinada por la composición de sus ácidos grasos polinsaturados y esto sugiere una nueva estrategia para la terapia antifúngica, que involucra tratamientos que incrementan la fluidez de la membrana plasmática para hacer el hongo susceptible a biocompuestos como el quitosano.

El quitosano también actúa como agente quelante que une, selectivamente, a trazas de metales y, por consiguiente, inhibe la producción de toxinas y el crecimiento micelial (5454. Cuero RG, Duffus E, Osuji G, Pettit R. Aflatoxin control in preharvest maize: effects of chitosan and two microbial agents. The Journal of Agricultural Science [Internet]. 1991;117(2):165-9. Available from: https://www.cambridge.org/core/journals/journal-of-agricultural- science/article/abs/aflatoxin-control-in-preharvest-maize-effects-of-chitosan-and-two-microbial- agents/BD8D14313D49649D683B711CE3F4DDE4 ). Además activa algunos procesos de defensa en los tejidos hospederos (5555. El Ghaouth A, Arul J, Asselin A, Benhamou N. Antifungal activity of chitosan on post- harvest pathogens: induction of morphological and cytological alterations in Rhizopus stolonifer. Mycological research [Internet]. 1992;96(9):769-79. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0953756209804474?via%3Dihub ) e inhibe varias enzimas. La unión del quitosano con el ADN y la inhibición de la síntesis de ARNm y síntesis de proteínas (5656. Sudarshan NR, Hoover DG, Knorr D. Antibacterial action of chitosan. Food Biotechnology [Internet]. 1992;6(3):257-72. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0953756209804474?via%3Dihub ), provocando daños celulares y estructurales.

En el caso de las nanopartículas de plata (Ag) se basa en la posibilidad de que estás se adhieran y penetren en la membrana celular (5757. Avila-Quezada GD, Espino-Solis GP. Silver nanoparticles offer effective control of pathogenic bacteria in a wide range of food products. Pathogenic Bacteria [Internet]. 2019; Available from: https://www.intechopen.com/chapters/69239 ), causando desbalance osmótico en las esporas, siendo muy efectivas contra Magnaporthe grisea (5858. Jo Y-K, Kim BH, Jung G. Antifungal activity of silver ions and nanoparticles on phytopathogenic fungi. Plant disease [Internet]. 2009;93(10):1037-43. Available from: https://apsjournals.apsnet.org/doi/abs/10.1094/PDIS-93-10-1037 ).

Chitosan. Chemical and physical characteristics

Chitosan is a polysaccharide obtained from chitin, the second most abundant polysaccharide in nature. It is a linear copolymer formed by residues of D-glucosamine units to a greater extent and N-acetyl D-glucosamine to a lesser extent, randomly distributed and joined by β 1,4 bonds. According to the International Union of Pure and Applied Chemistry (IUPAC), it is 2-amino 2-deoxy-D-glucopyranose (D-glucosamine GlcN) and 2-acetamide-2-deoxy-D-glucopyranose N-acetyl glucosamine (11. Harish Prashanth KV, Tharanathan RN. Chitin/chitosan: modifications and their unlimited application potential-an overview. Trends in Food Science and Technology. 2007;18(3):117-31. https://doi.org/10.1016/j.tifs.2006.10.022 ). Both the content and sequence of these units will determine the physicochemical and biological properties of this polymer. Chitosan has a nitrogen (N) content greater than 7 and has a regular distribution of free amino groups, which can be protonated by certain acids, becoming positively charged and this gives it a polycationic character. This fact allows explaining some of its properties such as the ability to bind with negatively charged substances, such as lipids, proteins, colorants, among others; as well as flocculant, adherent and adsorbent, in addition to the typical reactions of amines (99. Muzzarelli RAA. Enzymatic synthesis of chitin and chitosan. Occurrence of chitin. In: Muzzarelli RAA, editor. Chitin [Internet]. Pergamon; 1977 [cited 28/08/2021]. p. 5-44. https://doi.org/10.1016/B978-0-08-020367-6.50007-6 ). When the amino groups of chitosan bind to the acid amino acids of proteins, electrostatic interactions are produced that lead to cellular disorders in microorganisms.

This biopolymer possesses antimicrobial activity against a wide variety of microorganisms including fungi, algae and some bacteria. Its functionality and activity depends on its characteristics such as: molecular mass, acetylationdegree, host cell, presence of natural nutrients, chemical or nutritional composition of substrates and environmental conditions. In this sense, with respect to acetylation degree, it can be stated that the lower the acetylation degree, the higher the antimicrobial activity (1010. Kong M, Chen X, Xue Y, Liu C, Yu L, Ji Q, et al. Preparation and antibacterial activity of chitosan microshperes in a solid dispersing system. Frontiers of Materials Science in China [Internet]. 2008;2(2):214-20. Available from: https://link.springer.com/content/pdf/10.1007/s11706-008-0036-2.pdf ,1111. Takahashi T, Imai M, Suzuki I, Sawai J. Growth inhibitory effect on bacteria of chitosan membranes regulated with deacetylation degree. Biochemical Engineering Journal [Internet]. 2008;40(3):485-91. Available from: https://www.sciencedirect.com/science/article/abs/pii/S1369703X08000430 ).

Pyriculariaoryzaecausal agent of rice blast or rice brusone

This plant pathogen is very efficient as it can reproduce sexually (teleomorph: Magnaporthegrisea Barr (It. Hebert) without Magnaportheoryzae) and asexually (anamorph: Pyriculariaoryzae). The Pyricularia/Magnaporthe Working Group has established under the auspices of the International Commission on Fungal Taxonomy (ICFT) the possibility of retaining the name Magnaporthe over Pyricularia. However, such conservation requires a change in the type species of the genus Magnaporthe and would cause numerous name changes for those species currently placed in Pyricularia (1212. Zhang N, Luo J, Rossman AY, Aoki T, Chuma I, Crous PW, et al. Generic names in Magnaporthales. IMA fungus [Internet]. 2016;7(1):155-9. https://doi.org/10.5598/imafungus.2016.07.01.09 ).

The asexually typed name Pyricularia is the correct name for the rice scorch fungus, which corresponds well with pathogenicity and ecological and evolutionary characteristics. Therefore, the name Pyriculariaoryzae should be used for the fungus that produces this disease. However, the synonym Magnaportheoryzae can continue to be referred to in publications as Pyriculariaoryzae (syn. Magnaportheoryzae). This practice will help to close a potential gap in the literature and knowledge of this important species (1212. Zhang N, Luo J, Rossman AY, Aoki T, Chuma I, Crous PW, et al. Generic names in Magnaporthales. IMA fungus [Internet]. 2016;7(1):155-9. https://doi.org/10.5598/imafungus.2016.07.01.09 ). As for grisea and oryzae, they are very different species. Grisea is for strains of Digitaria and oryzae for rice strains, wheat and other grasses; although in the literature they are considered synonyms and the four names are used: Pyriculariaoryzae, Magnaportheoryzae, Pyriculariagrisea and Magnaporthegrisea.

Pyriculariaoryzae, known as blast or brusone is one of the most serious diseases of rice (Oryzasativa L.), which has caused significant losses in yields worldwide (1313. Lugo L, Jayaro Y, González Á, Borges O. Identification of sources of partial resistance to Pyricularia grisea in rice cultivars and experimental lines. Fitopatología Venezolana [Internet]. 2008;21(2):51-8. Available from: https://www.cabdirect.org/cabdirect/abstract/20093204367 ).

The blast distribution is worldwide since it is found in all agroecosystems of the tropics and temperate zones where commercial rice is grown. Piriculariosis generates large losses in grain production, both in rainfed and irrigated systems. This disease has been reported in at least 85 countries worldwide. It was discovered in Italy in 1560 and it was later found in China (1637), Japan (1760), United States (1960), and India (1913) (1414. Rossman AY, Howard RJ, Valent B. Pyricularia grisea the correct name for the rice blast disease fungus. Mycologia [Internet]. 1990;82(4):509-12. Available from: https://www.tandfonline.com/doi/abs/10.1080/00275514.1990.12025916?journalCode=umyc20 ). Blast has caused significant losses, for example: in India 90 % (1515. Manjunatha B, Krishnappa M. Morphological characterization of Pyricularia oryzae causing blast disease in rice (Oryza sativa L.) from different zones of Karnataka. Journal of Pharmacognosy and Phytochemistry [Internet]. 2019;8(3):3749-53. Available from: https://www.phytojournal.com/archives/2019/vol8issue3/PartBC/8-3-279-550.pdf ), in China 70 % (1616. Ou SH. Rice diseases [Internet]. IRRI; 1985. Available from: https://books.google.es/books?hl=es&lr=&id=-k3mewv9nMoC&oi=fnd&pg=PR1&dq=Rice+Diseases&ots=Zmj_rBBc8i&sig=C413L03O2pB85A2EhfQhPD1KApU#v=onepage&q=Rice%20Diseases&f=false ), in Thailand only 1900 ha were affected in 1987, and in 1988 it increased to 490 000 ha, in Spain and in the Mediterranean area it has also caused damages (1717. Koutroubas SD, Katsantonis D, Ntanos DA, LUPOTTO E. Blast disease influence on agronomic and quality traits of rice varieties under Mediterranean conditions. Turkish Journal of Agriculture and forestry [Internet]. 2009;33(5):487-94. Available from: https://journals.tubitak.gov.tr/agriculture/abstract.htm?id=10484 ). Mexico, on the other hand, has reported a drop in production of up to 30 % in rainfed crops. Cuba has been another country where this pathogen has caused damage, and when conditions are favorable, losses have increased up to 70 % (66. Rodríguez AT, Ramírez MA, Cárdenas RM, Hernández AN, Velázquez MG, Bautista S. Induction of defense response of Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating seeds with chitosan and hydrolyzed chitosan. Pesticide Biochemistry and Physiology [Internet]. 2007;89(3):206-15. https://doi.org/10.1016/j.pestbp.2007.06.007 ).

Taxonomic classification, morphology and symptomatology of Pyriculariaoryzae

The causal agent of blast is taxonomically classified in the class: Deuteromycetes, order: Moniliales, family: Dematiaceae, genus: Pyricularia, species: Pyriculariaoryzae. Pyriculariaoryzae has simple, partitioned, brownish conidiophores (Figure 1A). The conidiophores are borne singly or in groups of three and carry conidia at their ends. The conidiophores are solitary or in groups of three and at their ends carry the conidia, which are hyaline, fusiform and divided by two equidistant septa.

It is a complex disease due to the pathogenic variability and the rapidity with which this fungus overcomes the resistance of the rice plant. The mycelium of the fungus produces a toxic substance known as pyricularin (1616. Ou SH. Rice diseases [Internet]. IRRI; 1985. Available from: https://books.google.es/books?hl=es&lr=&id=-k3mewv9nMoC&oi=fnd&pg=PR1&dq=Rice+Diseases&ots=Zmj_rBBc8i&sig=C413L03O2pB85A2EhfQhPD1KApU#v=onepage&q=Rice%20Diseases&f=false ), which inhibits the growth of tissues and disorganizes them. It attacks the aerial parts of plant such as leaves, stems, nodes and spikes (1818. Kulmitra AK, Sahu N, Sahu MK, Kumar R, Kushram T, Sanath Kumar VB. Growth of Rice blast fungus Pyricularia oryzae (Cav.) on different solid and liquid media. International Journal of Current Microbiology and Applied Sciences [Internet]. 2017;6(6):1154-60. https://doi.org/10.20546/ijcmas.2017.606.133 ) (Figures 1B,C). The fungus produces spots or lesions on leaves of elongated or elliptical to rhomboid shape and uniform brown color that later will change to a grayish color in the central part, a fact that indicates the sporulation of the fungus; although its size and color vary according to environmental conditions and the susceptibility of the varieties (1919. Cárdenas RM, Pérez N, Cristo E, González MC, Fabré L. Estudio sobre el comportamiento de líneas y variedades de arroz (Oryza sativa Lin.) ante la infección por el hongo Pyricularia grisea Sacc. Cultivos Tropicales [Internet]. 2005;26(4):83-7. Available from: https://www.redalyc.org/pdf/1932/193216160012.pdf ). Its optimum growth temperature is between 22-29 ºC and a high relative humidity around 90 %, which fully coincide with the climatic conditions of tropical countries. The presence of high concentrations of nitrogen in the water also favors the development of fungus and produces large quantities of spores. The spores arrive from the remains of the previous season's harvest or from weeds where the fungus has been lodged during the winter (2020. Cárdenas RM, Polón CR, Pérez N, Cristo E, Mesa S, Fabré L, et al. Relación entre la incidencia de la piriculariosis (Pyricularia grisea Sacc.) del arroz (Oryza sativa Lin.) y diferentes variables climáticas en el Complejo Agroindustrial Arrocero Los Palacios. Cultivos Tropicales [Internet]. 2010;31(1):00-00. Available from: http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S0258-59362010000100002 ).

General strategies for the Pyriculariaoryzae control

Disease control is mainly based on the application of systemic chemical fungicides, including benzimidazole fungicides such as prochloraz, tebuconazole and propiconazole, among others. However, their use has had several disadvantages, since they cause contamination of the water table and adjacent bodies of water, generating harmful effects on various organisms. In addition, it is important to point out that these chemical fungicides are being damaged by the fungus, due to the emergence of populations with loss of sensitivity to their mode of action (2121. Manzo Sánchez G, Carrillo Madrigal H, Guzmán González S, Orozco Santos M. Análisis de la Sensibilidad in vitro de Mycosphaerella fijiensis, Agente Causal de la Sigatoka Negra del Banano a los Fungicidas Benomyl, Propiconazol y Azoxistrobin. Revista mexicana de fitopatología [Internet]. 2012 [cited 28/08/2021];30(1):81-5. Available from: http://www.scielo.org.mx/scielo.php?script=sci_abstract&pid=S0185-33092012000100008&lng=es&nrm=iso&tlng=es ). Due to this situation, there is an increasing number of fungicide cycles per year to protect crops against this disease, which implies an increase in production costs and in negative effects of conventional fungicides on the environment, questioning their commercial use, being then a priority the search for alternatives that allow complementing the integrated management of diseases.

In vitro and in vivo activity of chitosan and its derivatives on Pyriculariaoryzae

The antimicrobial property of chitosan and its derivatives have received considerable attention in recent years due to the impending problem associated with synthetic chemical agents. These compounds have been shown to be fungicidal and fungistatic for the control of Botrytis cinerea, Aspergillus flavus, Aspergillus parasiticus, Drechstera sorokiana, Fusarium acuminatum, Fusarium graminearum, Micronectriella nivalis, Rhizoctonia solani, Alternaria alternata, Colletotrichum gloesporioides, Penicillumspp, Fusarium oxysporum and Bipolaris oryzae, among other fungi (2222. Hua C, Li Y, Wang X, Kai K, Su M, Zhang D, et al. The effect of low and high molecular weight chitosan on the control of gray mold (Botrytis cinerea) on kiwifruit and host response. Scientia Horticulturae [Internet]. 2019;246:700-9. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0304423818308197 -2626. Rodríguez Pedroso AT, Plascencia Jatomea M, Bautista Baños S, Cortez Rocha MO, Ramírez Arrebato MÁ. Actividad antifúngica in vitro de quitosanos sobre Bipolaris oryzae patógeno del arroz. Acta Agronómica [Internet]. 2016;65(1):98-103. Available from: http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0120-28122016000100014 ).

In this regard, the antifungal chitosan activity and its derivatives has been observed in different stages of fungal development, such as affecting the growth and development of the pathogen, sporulation, viability and germination of spores and the production of fungal virulence factors (2323. Sánchez-Domínguez D, Ríos MY, Castillo-Ocampo P, Zavala-Padilla G, Ramos-García M, Bautista-Baños S. Cytological and biochemical changes induced by chitosan in the pathosystem Alternaria alternata-tomato. Pesticide biochemistry and physiology [Internet]. 2011;99(3):250-5. Available from: https://d1wqtxts1xzle7.cloudfront.net/44367734/Cytological_and_biochemical_changes_indu20160403-12579-18vumyq.pdf?1459730343=&response-content-disposition=inline%3B+filename%3DCytological_and_biochemical_changes_indu.pdf&Expires=1631769785&Signature=IGGgjxkuWLCyHKJnBKWJkxGt1dhC3jDsalPwe033Zh69KP~oMc9leISiFCA0WSiKUCxKb1K~RCJkP0URuU~09UxsZWRx6yjMBZ-~tCQYVGBeFq4iuV~0LuAXIeh1ysKZ53HVKZb0Z2fVsYsTRC~FMcGsjqZbRwVPD1mjM7VE43h3RvolAkQ4LLzRWs1KSSscUcUHoy-8cVMHx~zmJFU7DCf~ZGlzwERIsr7TgSW2w7xT7vzMf-OaiiFMda~v241ukzc5Vd6HBXZUxr1J16f266xhUCV5w6uDBnm4X3MHMaFFjIaf9XPSJdfnmDnR8TAzi11jPKfVHXjjuNPU0Y8Zg&Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA ,2727. Živković S, Stevanović M, Đurović S, Ristić D, Stošić S. Antifungal activity of chitosan against Alternaria alternata and Colletotrichum gloeosporioides. Pesticidi i fitomedicina [Internet]. 2018;33(3-4):197-204. Available from: https://plantarum.izbis.bg.ac.rs/bitstream/handle/123456789/543/541.pdf?sequence=1 ,2828. Badawy ME, Rabea EI. A biopolymer chitosan and its derivatives as promising antimicrobial agents against plant pathogens and their applications in crop protection. International Journal of Carbohydrate Chemistry [Internet]. 2011;2011. https://doi.org/10.1155/2011/460381 ). Some authors have verified the fungicidal effect of these compounds at different concentrations and P. griseaisolates (55. Rodríguez AT, Ramírez MA, Nápoles MC, Márquez R, Cárdenas RM. Antifungal activity of chitosan and one of its hydrolysates on Pyricularia grisea, Sacc. Fungus. Cultivos Tropicales [Internet]. 2003;24(2):85-8. Available from: https://www.redalyc.org/pdf/1932/193218174015.pdf ,77. Cárdenas RM, Ramírez MA, Rodríguez AT, González LM. Efecto de los derivados de quitina y su combinación con sulfato de cobre en el comportamiento del crecimiento micelial y esporulación de un aislamiento monospórico del hongo Pyricularia grisea Sacc. Cultivos Tropicales [Internet]. 2004;25(4):89-93. Available from: https://www.redalyc.org/pdf/1932/193225911012.pdf ,2929. Rabea EI, Badawy ME, Rogge TM, Stevens CV, Höfte M, Steurbaut W, et al. Insecticidal and fungicidal activity of new synthesized chitosan derivatives. Pest Management Science [Internet]. 2005;61(10):951-60. https://doi.org/10.1002/ps.1085 ). Chitosan oligomers have also been shown to have a better inhibitory effect on this pathogen, achieving the minimum inhibitory concentration at a concentration higher than 2000 mg L^-1 (3030. Xu J, Zhao X, Han X, Du Y. Antifungal activity of oligochitosan against Phytophthora capsici and other plant pathogenic fungi in vitro. Pesticide Biochemistry and Physiology. 2007;87(3):220-8.). Studies carried out in the laboratory of Oligosaccharins of the National Institute of Agricultural Sciences (INCA) with P. grisea indicate that chitosan and its oligomers in the culture medium at a concentration of 1,000 mg L^-1 and pH 5.6, totally inhibited the mycelial growth of this fungus (55. Rodríguez AT, Ramírez MA, Nápoles MC, Márquez R, Cárdenas RM. Antifungal activity of chitosan and one of its hydrolysates on Pyricularia grisea, Sacc. Fungus. Cultivos Tropicales [Internet]. 2003;24(2):85-8. Available from: https://www.redalyc.org/pdf/1932/193218174015.pdf ). However, it is important to consider the pH of the resulting solution, which affects the positive charge of the amino groups, since in another test at pH 6 there was only a slight affectation of the fungus growth, although a total inhibition of sporulation was maintained (77. Cárdenas RM, Ramírez MA, Rodríguez AT, González LM. Efecto de los derivados de quitina y su combinación con sulfato de cobre en el comportamiento del crecimiento micelial y esporulación de un aislamiento monospórico del hongo Pyricularia grisea Sacc. Cultivos Tropicales [Internet]. 2004;25(4):89-93. Available from: https://www.redalyc.org/pdf/1932/193225911012.pdf ).

Some research groups have begun to modify the chitosan molecule with the addition of hydrophobic groups to increase its biological activity against this pathogen. For example, N-sulfonated N-sulfobenzoyl chitosan (3131. Chen C-S, Liau W-Y, Tsai G-J. Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation. Journal of Food Protection [Internet]. 1998;61(9):1124-8. https://doi.org/10.4315/0362-028X-61.9.1124 ), N,N,N. trimethyl chitosan (3232. Jia Z, Xu W. Synthesis and antibacterial activities of quaternary ammonium salt of chitosan. Carbohydrate research [Internet]. 2001;333(1):1-6. https://doi.org/10.1016/S0008-6215(01)00112-4 ) N,O-acyl chitosan (3333. Sashiwa H, Kawasaki N, Nakayama A, Muraki E, Yamamoto N, Zhu H, et al. Chemical modification of chitosan. Synthesis of organosoluble, palladium adsorbable, and biodegradable chitosan derivatives toward the chemical plating on plastics. Biomacromolecules [Internet]. 2002;3(5):1120-5. https://doi.org/10.1021/bm0200478 ), O-acyl chitosan (3434. Badawy ME, Rabea EI, Rogge TM, Stevens CV, Steurbaut W, Höfte M, et al. Fungicidal and insecticidal activity of O-acyl chitosan derivatives. Polymer bulletin. 2005;54(4):279-89. https://doi.org/10.1007/s00289-005-0396-z ,3535. Badawy M, Rabea E, Steurbaut W, Rogge T, Stevens C, Smagghe G, et al. Fungicidal activity of some O-acyl chitosan derivatives against grey mould Botrytis cinerea and rice leaf blast Pyricularia grisea. Communications in agricultural and applied biological sciences [Internet]. 2005;70(3):215-8. Available from: https://europepmc.org/article/med/16637180 ), hydroxyethyl aryl chitosan (3636. Ma G, Yang D, Tan H, Wu Q, Nie J. Preparation and characterization of N‐alkylated chitosan derivatives. Journal of applied polymer science [Internet]. 2008;109(2):1093-8. https://doi.org/10.1002/app.28223 ), dimethylpiperazine chitosan (3737. Másson M, Holappa J, Hjálmarsdóttir M, Rúnarsson ÖV, Nevalainen T, Järvinen T. Antimicrobial activity of piperazine derivatives of chitosan. Carbohydrate polymers [Internet]. 2008;74(3):566-71. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0144861708001720 ), carboxymethyl chitosan (3838. Seyfarth F, Schliemann S, Elsner P, Hipler U-C. Antifungal effect of high-and low- molecular-weight chitosan hydrochloride, carboxymethyl chitosan, chitosan oligosaccharide and N- acetyl-D-glucosamine against Candida albicans, Candida krusei and Candida glabrata. International Journal of Pharmaceutics [Internet]. 2008;353(1-2):139-48. Available from: https://pubmed.ncbi.nlm.nih.gov/18164151/ ), acyl urea thiourea chitosan (3939. Zhong Z, Xing R, Liu S, Wang L, Cai S, Li P. Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro. Carbohydrate Research [Internet]. 2008;343(3):566-70. Available from: https://www.researchgate.net/profile/Shengbao-Cai/publication/5765859_Synthesis_of_acyl_thiourea_derivatives_of_chitosan_and_their_antimicrobial_activities_in_vitro/links/5fd7458745851553a0b591d5/Synthesis-of-acyl-thiourea-derivatives-of-chitosan-and-their-antimicrobial-activities-in-vitro.pdf ), N-succinoyl chitosan (4040. Tikhonov VE, Stepnova EA, Babak VG, Yamskov IA, Palma-Guerrero J, Jansson H-B, et al. Bactericidal and antifungal activities of a low molecular weight chitosan and its N-/2 (3)-(dodec- 2-enyl) succinoyl/-derivatives. Carbohydrate polymers. 2006;64(1):66-72.) and N-heterocyclic chitosan (4141. Stössel P, Leuba JL. Effect of chitosan, chitin and some aminosugars on growth of various soilborne phytopathogenic fungi. Journal of Phytopathology [Internet]. 1984;111(1):82-90. Available from: https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1439-0434.1984.tb04244.x ). Researchers have noted that N-alkylation or N-arylation of chitosan with aromatic or aliphatic aldehydes effectively increased its antifungal activity on P. grisea (4242. Badawy ME, Rabea EI. Characterization and antimicrobial activity of water-soluble N-(4- carboxybutyroyl) chitosans against some plant pathogenic bacteria and fungi. Carbohydrate polymers [Internet]. 2012;87(1):250-6. Available from: http://damanhour.edu.eg/pdf/researches/1-s2.0-S014486171100645X-main.pdf ).

With the same methods and obtaining techniques, but using different types of aldehydes, other scientists (3131. Chen C-S, Liau W-Y, Tsai G-J. Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation. Journal of Food Protection [Internet]. 1998;61(9):1124-8. https://doi.org/10.4315/0362-028X-61.9.1124 ), observed antifungal activity of 24 new chitosan derivatives (N-benzyl chitosan derivatives), which had a greater inhibitory effect than native chitosans on the growth and spore formation of P. grisea, with N-(m-nitrobenzyl) chitosan having the greatest effect at a concentration of 5 g L^-1. These authors also observed that the most active derivative was N-(2,2diphenylethyl) chitosan, with a minimum inhibitory concentration of 0.3 g L^-1 against this pathogen. On the other hand, great advances of chitosan and its oligomers on the direct control of rice diseases have been observed. Both chitin and chitosan have been shown to induce the accumulation of phytoalexin production in this case of momilactones A and momilactones B upon infection with P.grisea in rice leaves at a concentration of 10 µg mL^-1 (4343. Shimizu T, Jikumaru Y, Okada A, Okada K, Koga J, Umemura K, et al. Effects of a bile acid elicitor, cholic acid, on the biosynthesis of diterpenoid phytoalexins in suspension-cultured rice cells. Phytochemistry [Internet]. 2008;69(4):973-81. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0031942207006097?via%3Dihub ).

A group of researchers published the chitosan effect in stimulating defense responses in rice leaves (4444. Agrawal GK, Rakwal R, Tamogami S, Yonekura M, Kubo A, Saji H. Chitosan activates defense/stress response (s) in the leaves of Oryza sativa seedlings. Plant Physiology and Biochemistry. 2002;40(12):1061-9.). After treatment with 0.1 % chitosan, necrosis was clearly observed in the upper part of rice leaf. However, treating rice seedlings with 5 mg L^-1 and inoculating them with Magnaporthegrisea 97-23-2D1 showed a better effect and control of the disease by more than 50 % (4545. Lin W, Hu X, Zhang W, Rogers WJ, Cai W. Hydrogen peroxide mediates defence responses induced by chitosans of different molecular weights in rice. Journal of Plant Physiology [Internet]. 2005;162(8):937-44. Available from: http://sippe.ac.cn/gh/papers/caiweiming/Lin2005.pdf ). However, in 2007 it was evaluated, in semi-controlled conditions, where rice seeds were treated at different concentrations with two chitosans of different molecular weight (66. Rodríguez AT, Ramírez MA, Cárdenas RM, Hernández AN, Velázquez MG, Bautista S. Induction of defense response of Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating seeds with chitosan and hydrolyzed chitosan. Pesticide Biochemistry and Physiology [Internet]. 2007;89(3):206-15. https://doi.org/10.1016/j.pestbp.2007.06.007 ). Eighteen days after seed germination, plants obtained were inoculated with spores of P. grisea, and the activity of enzymes related to defense such as PAL, glucanase, chitinase and chitosanase was determined, observing an increase in the activity of plants treated with elicitors, in relation to the control. In addition, no disease symptoms were observed in the highest concentration of both compounds used (66. Rodríguez AT, Ramírez MA, Cárdenas RM, Hernández AN, Velázquez MG, Bautista S. Induction of defense response of Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating seeds with chitosan and hydrolyzed chitosan. Pesticide Biochemistry and Physiology [Internet]. 2007;89(3):206-15. https://doi.org/10.1016/j.pestbp.2007.06.007 ).

Currently, research is being conducted on the application of chitosan-based nanoparticles with antifungal activity and for the control of diseases such as pyriculariosis (88. Manikandan A, Sathiyabama M. Preparation of chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea. International journal of biological macromolecules [Internet]. 2016;84:58-61. https://doi.org/10. 1016/j.ijbiomac.2015.11.083 ,4646. Pham DC, Nguyen TH, Ngoc UTP, Le NTT, Tran TV, Nguyen DH. Preparation, characterization and antifungal properties of chitosan-silver nanoparticles synergize fungicide against Pyricularia oryzae. Journal of nanoscience and nanotechnology [Internet]. 2018;18(8):5299-305. Available from: https://www.researchgate.net/profile/Dinh-Chuong-Pham/publication/321308729_Preparation_Characterization_and_Antifungal_Properties_of_Chitosan-Silver_Nanoparticles_Synergize_Fungicide-Against-Pyricularia-oryzae/links/5a35486245851532e82f1da0/Preparation-Characterization-and-Antifungal-Properties-of-Chitosan-Silver-Nanoparticles-Synergize-Fungicide-Against-Pyricularia-oryzae.pdf ,4747. Nguyen TH, Thi TV, Nguyen T-T, Le TD, Vo DMH, Nguyen DH, et al. Investigation of chitosan nanoparticles loaded with protocatechuic acid (PCA) for the resistance of Pyricularia oryzae fungus against rice blast. Polymers. 2019;11(1):177.). On the other hand, researchers found that chitosan and silver (Ag) nanoparticles had a high antifungal activity on Pyriculariaoryzae at anAgconcentration (2 ppm) and chitosan (4000 ppm) (4646. Pham DC, Nguyen TH, Ngoc UTP, Le NTT, Tran TV, Nguyen DH. Preparation, characterization and antifungal properties of chitosan-silver nanoparticles synergize fungicide against Pyricularia oryzae. Journal of nanoscience and nanotechnology [Internet]. 2018;18(8):5299-305. Available from: https://www.researchgate.net/profile/Dinh-Chuong-Pham/publication/321308729_Preparation_Characterization_and_Antifungal_Properties_of_Chitosan-Silver_Nanoparticles_Synergize_Fungicide-Against-Pyricularia-oryzae/links/5a35486245851532e82f1da0/Preparation-Characterization-and-Antifungal-Properties-of-Chitosan-Silver-Nanoparticles-Synergize-Fungicide-Against-Pyricularia-oryzae.pdf ). However, other scientists observed that applying 500 μl of a 0.1 % chitosan nanoparticle solution on rice leaves and 24 h later inoculated a spore suspension (1x10⁵spores mL^-1) of P. grisea (88. Manikandan A, Sathiyabama M. Preparation of chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea. International journal of biological macromolecules [Internet]. 2016;84:58-61. https://doi.org/10. 1016/j.ijbiomac.2015.11.083 ). After 10 days, no symptoms of the disease were observed; this could be due to the stimulation of some defense mechanism in leaves and controlled the infection (88. Manikandan A, Sathiyabama M. Preparation of chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea. International journal of biological macromolecules [Internet]. 2016;84:58-61. https://doi.org/10. 1016/j.ijbiomac.2015.11.083 ).

Chitosan action mechanisms

Chitosan action mechanisms have not been fully established, although there are some hypotheses in this regard. In general, the various proposals to explain the antimicrobial activity of chitosan consider as a fundamental characteristic the polycationic nature of the molecule, which is given by the NH³⁺ groups of glucosamine, which confers important biological and physiological properties (4848. Je J-Y, Kim S-K. Antimicrobial action of novel chitin derivative. Biochimica et Biophysica Acta (BBA)-General Subjects [Internet]. 2006;1760(1):104-9. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0304416505003028?via%3Dihub ,4949. El Hadrami A, Adam LR, El Hadrami I, Daayf F. Chitosan in plant protection. Marine drugs. 2010;8(4):968-87.). In pH conditions, chitosan behaves as a linear polyelectrolyte, with a pK around 6.5, therefore at low pH the glucosamine residues are positively charged, due to the protonation of its amino residues, containing a high density of positive charges, which allows it to bind strongly to negatively charged surfaces (5050. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM. Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryotic Cell [Internet]. 2005;4(4):703-15. Available from: https://journals.asm.org/doi/pdf/10.1128/EC.4.4.703-715.2005 ). It is proposed that when the positive charge on the C-2 of the glucosamine monomer is below pH 6, chitosan is more soluble and has better antimicrobial activity than chitin (3131. Chen C-S, Liau W-Y, Tsai G-J. Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation. Journal of Food Protection [Internet]. 1998;61(9):1124-8. https://doi.org/10.4315/0362-028X-61.9.1124 ,5151. Velásquez CL. Some potentialities of chitin and chitosan for uses related to agriculture in Latin America. Revista Científica UDO Agrícola. 2008;8(1):1-22.).

Another proposed mechanism is the interaction between the positive charge of the chitosan molecule and the negative charge of the microbial membrane cells leading to the efflux of proteins and other intracellular constituents (3131. Chen C-S, Liau W-Y, Tsai G-J. Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation. Journal of Food Protection [Internet]. 1998;61(9):1124-8. https://doi.org/10.4315/0362-028X-61.9.1124 ). The most abundant of the sphingolipids is mannosyldiinositrophosphate-ceramide (M(IP2)C), which has two negative charges. These negative charges corresponding to the plasma membrane (M(IP2)C) can bind to the amino groups of the glucosamine residues of chitosan. In other studies, it has been observed that chitosan forms transport channels for molecules in artificial lipid bilayers, which provides evidence that this compound can disorganize the cell membrane (5050. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM. Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryotic Cell [Internet]. 2005;4(4):703-15. Available from: https://journals.asm.org/doi/pdf/10.1128/EC.4.4.703-715.2005 ). A working group analyzed the chitosan action mode on fungal cells and observed two aspects: that chitosan permeabilizes the fungal plasma membrane and penetrates fungal cells, a process that is ATP-dependent, and also demonstrated that different cell types (conidium, germ tube and hyphae) exhibit different sensitivity to chitosan (5252. Palma-Guerrero J, Huang I-C, Jansson H-B, Salinas J, Lopez-Llorca LV, Read ND. Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner. Fungal Genetics and Biology [Internet]. 2009;46(8):585-94. Available from: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.458.5391&rep=rep1&type=pdf ,5353. López Jiménez JÁ, Palma Guerrero J, Pérez Berná AJ, Huang IC, Jansson HB, Salinas J, et al. Membrane fluidity determines sensitivity of filamentous fungi to chitosan. Molecular Microbiology, n^o75, vol. 4 [Internet]. 2010; Available from: https://digitum.um.es/digitum/bitstream/10201/38147/1/Membrane%20fluidity%20determines%20sensitivity.pdf ). In 2010, the same group of researchers demonstrated, through biological, biochemical, genetic and biophysical techniques, that the antifungal activity of chitosan depends on the fluidity of the fungal plasma membrane, which is determined by the composition of its polyunsaturated fatty acids, and this suggests a new strategy for antifungal therapy, involving treatments that increase the plasma membrane fluidity to make the fungus susceptible to biocompounds such as chitosan.

Chitosan also acts as a chelating agent that selectively binds trace metals and thereby inhibits toxin production and mycelial growth (5454. Cuero RG, Duffus E, Osuji G, Pettit R. Aflatoxin control in preharvest maize: effects of chitosan and two microbial agents. The Journal of Agricultural Science [Internet]. 1991;117(2):165-9. Available from: https://www.cambridge.org/core/journals/journal-of-agricultural- science/article/abs/aflatoxin-control-in-preharvest-maize-effects-of-chitosan-and-two-microbial- agents/BD8D14313D49649D683B711CE3F4DDE4 ). It also activates some defense processes in host tissues (5555. El Ghaouth A, Arul J, Asselin A, Benhamou N. Antifungal activity of chitosan on post- harvest pathogens: induction of morphological and cytological alterations in Rhizopus stolonifer. Mycological research [Internet]. 1992;96(9):769-79. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0953756209804474?via%3Dihub ) and inhibits several enzymes. The binding of chitosan to DNA and the inhibition of mRNA synthesis and protein synthesis (5656. Sudarshan NR, Hoover DG, Knorr D. Antibacterial action of chitosan. Food Biotechnology [Internet]. 1992;6(3):257-72. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0953756209804474?via%3Dihub ), causing cellular and structural damage.

In the case of silver nanoparticles (Ag), it is based on the possibility that they adhere and penetrate the cell membrane (5757. Avila-Quezada GD, Espino-Solis GP. Silver nanoparticles offer effective control of pathogenic bacteria in a wide range of food products. Pathogenic Bacteria [Internet]. 2019; Available from: https://www.intechopen.com/chapters/69239 ), causing osmotic imbalance in spores, being very effective against Magnaporthegrisea (5858. Jo Y-K, Kim BH, Jung G. Antifungal activity of silver ions and nanoparticles on phytopathogenic fungi. Plant disease [Internet]. 2009;93(10):1037-43. Available from: https://apsjournals.apsnet.org/doi/abs/10.1094/PDIS-93-10-1037 ).