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 lineal 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. Prashanth KV, Tharanathan RN. Chitin/chitosan: modifications and their unlimited application potential-an overview. Trends in Food Science & Technology. 2007;18(3):117-31. doi: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 alto contenido de nitrógeno (N) 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. Chitin. 1977;5-17.). 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.

Pyricularia oryzae, agente causal del añublo o quemazón del arroz

Pyricularia y Magnaporthe son, actualmente, nombres genéricos ampliamente utilizados, para el hongo que produce la quemazón del arroz. El Grupo de Trabajo Pyricularia/ Magnaporthe ha considerado la posibilidad de conservar el nombre Magnaporthe sobre Pyricularia. Sin embargo, dicha conservación requeriría un cambio en la especie tipo del género Magnaporthe y causaría numerosos cambios de nombre para aquellas especies actualmente ubicadas en Pyricularia.

El nombre genérico 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 (1010. Zhang N, Luo J, Rossman AY, Aoki T, Chuma I, Crous PW, et al. Generic names in Magnaporthales. IMA fungus. 2016;7(1):155-9.). 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 (1111. 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. 2008;21(2):51-8.). Se trata de un patógeno de plantas muy eficaz, ya que pueden reproducirse sexualmente (teleomorfo: Magnaporthe grisea Barr (It. Hebert) sin. Magnaporthe oryzae) y asexualmente (anamorfo: Pyricularia oryzae). 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) (1212. Rossman AY, Howard RJ, Valent B. Pyricularia grisea the correct name for the rice blast disease fungus. Mycologia. 1990;82(4):509-12.). La piriculariosis ha causado significantes pérdidas, por ejemplo: en la India el 90 % (1313. 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. 2019;8(3):3749-53.), en China el 70 % (1414. Ou SH. Rice Diseases: Commonwealth Mycological Institute. 2nd ed. Kew Surrey, England; 1985. 380 p.), en Tailandia fue afectada en 1987 solamente un área de 1900 ha y ya en 1988 se incrementó a 490 000 ha, en España y en la zona del mediterráneo también ha causado daños (1515. 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. 2009;33(5):487-94.). 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 que ha sido afectado por este patógeno, y cuando las condiciones son favorables se ha incrementado las pérdidas hasta un 70 %.

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. Estos hialinos, fusiformes y están divididos en forma equidistante por dos septos. Sus medidas aproximadas son 22 a 24 µm x 10 a 12 µm. Más de un conidio puede formarse sobre el conidióforo, el número de conidios oscila entre 1 y 20.

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, que inhibe el crecimiento de los tejidos y los desorganiza. Ataca las partes aéreas de la planta como las hojas, tallos, nudos y espigas (1616. 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. 2017;6(6):1154-60.) (Figuras 1B, C). El hongo produce unas manchas o lesiones en las hojas de forma alargada o elíptica a romboide, 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 (1717. 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. 2005;26(4):83-7.). Su temperatura óptima de crecimiento está entre 22-29 ^oC y una elevada humedad relativa, entorno al 90 %, las 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 (1818. 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. 2010;31(1):14-8.).

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 (1919. Patiño L. La resistencia a fungicidas, una continúa amenaza al control de la Sigatoka Negra. Boletín Técnico Cenibanano. 2003;4:2-5.). 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, cuestionandose su uso comercial, siendo prioritaria 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 ha 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 (2020. 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. 2019;246:700-9.-2424. Rodríguez Pedroso AT, Plascencia Jatomea M, Bautista Baños S, Cortez Rocha MO, Ramírez Arrebato MÁ. Actividad antifúngica in vitro de quitosanos sobre patógeno del arroz. Acta Agronomica. 2016;65(2):169-74.).

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 (2121. 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. 2011;99(3):250-5.,2525. Živković S, Stevanović M, Đurović S, Ristić D, Stošić S. Antifungal activity of chitosan against Alternaria alternata and Colletotrichum gloeosporioides. Pesticidi i fitomedicina. 2018;33(3-4):197-204.,2626. 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. 2011;2011.). Algunos autores han demostrado 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. 2003;24(2):85-8.,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. 2004;25(4):89-93.,2727. 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. 2005;61(10):951-60.). 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. (2828. 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. 2003;24(2):85-8.). 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. 2004;25(4):89-93.)

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 (2929. 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. 1998;61(9):1124-8.), N,N,N.trimetil quitosano (3030. Jia Z, Xu W. Synthesis and antibacterial activities of quaternary ammonium salt of chitosan. Carbohydrate research. 2001;333(1):1-6.), N,O-acyl quitosano (3131. Sashiwa H, Kawasaki N, Nakayama A, Muraki E, Yamamoto N, Zhu H, et al. Chemical modification of chitosan. 13. Synthesis of organosoluble, palladium adsorbable, and biodegradable chitosan derivatives toward the chemical plating on plastics. Biomacromolecules. 2002;3(5):1120-5.), O-acil quitosano (3232. 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.,3333. 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. 2005;70(3):215-8.), hidroxietil aril quitosano (3434. Ma G, Yang D, Tan H, Wu Q, Nie J. Preparation and characterization of N‐alkylated chitosan derivatives. Journal of applied polymer science. 2008;109(2):1093-8.), dimetilpiperazina quitosano (3535. Másson M, Holappa J, Hjálmarsdóttir M, Rúnarsson ÖV, Nevalainen T, Järvinen T. Antimicrobial activity of piperazine derivatives of chitosan. Carbohydrate polymers. 2008;74(3):566-71.), carboximetil quitosano (3636. 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. 2008;353(1-2):139-48.), acil urea tiourea quitosano (3737. 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. 2008;343(3):566-70.), N-succinoil quitosano (3838. 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 (3939. Stössel P, Leuba JL. Effect of chitosan, chitin and some aminosugars on growth of various soilborne phytopathogenic fungi. Journal of Phytopathology. 1984;111(1):82-90.). Investigadores (4040. Badawy ME, Rabea EI. Characterization and antimicrobial activity of water-soluble N-(4-carboxybutyroyl) chitosans against some plant pathogenic bacteria and fungi. Carbohydrate polymers. 2012;87(1):250-6.) han notado que la N-alquilación o N-arilación del quitosano con aldehídos aromáticos o alifáticos aumentaron, efectivamente, su actividad antifúngica sobre P. grisea.

Con los mismos métodos y técnicas de obtención, pero utilizando diferentes tipos de aldehídos, científicos (2929. 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. 1998;61(9):1124-8.) 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 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 (4141. 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. 2008;69(4):973-81.) y momilactonas B ante una infección con P. grisea en hojas de arroz a la concentración de 10 µg mL^-1.

Un grupo de investigadores (4242. 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.) publicó el efecto del quitosano en la estimulación de respuestas de defensa en hojas de arroz. 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 % (4343. 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. 2005;162(8):937-44.). 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. 2007;89(3):206-15.). A los 18 días de germinada la semilla, las plantas obtenidas fueron inoculadas con esporas de P. grisea, se evaluó 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, con respecto al control. Además, no se observaron síntomas de la enfermedad en la concentración más elevada utilizada, de ambos compuestos.

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. 2016;84:58-61.,4444. 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.,4545. 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. 2018;18(8):5299-305.). Por su parte, estudiosos del tema (4444. 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.) 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). Sin embargo, otros científicos (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. 2016;84:58-61.) aplicaron 500 μl de una solución de nanopartículas de quitosano al 0,1 % sobre hojas de arroz, y 24 h más tarde, una suspensión de esporas (1x10⁵ esporas mL^-1) de P. grisea, y a los 10 días, no observaron síntomas de la enfermedad.

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 (4646. Je J-Y, Kim S-K. Antimicrobial action of novel chitin derivative. Biochimica et Biophysica Acta (BBA)-General Subjects. 2006;1760(1):104-9.,4747. 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 pH 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 (4848. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM. Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryotic Cell. 2005;4(4):703-15.). Se plantea que cuando la carga positiva sobre el C-2 del monómero de glucosamina se encuentra por debajo de pH 6, el quitosano es más soluble y tiene una mejor actividad antimicrobiana que la quitina (2929. 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. 1998;61(9):1124-8.,4949. 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, o que conduce a la salida de proteínas y otros constituyentes intracelulares (2929. 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. 1998;61(9):1124-8.). El más abundante de los esfingolípidos es el manosildiinositolfosfato-ceramida (M(IP₂) C), el cual presenta dos cargas negativas. Sitios donde el quitosano podría unirse utilizando sus grupos amino de los residuos de glucosamina, los cuales son capaces de interactuar con los componentes negativos del (M(IP₂) C) de la membrana plasmática. 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 (4848. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM. Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryotic Cell. 2005;4(4):703-15.). Por su parte, un grupo de trabajo (5050. 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. 2009;46(8):585-94.,5151. Palma‐Guerrero J, Lopez‐Jimenez JA, Pérez‐Berná AJ, Huang I-C, Jansson H-B, Salinas J, et al. Membrane fluidity determines sensitivity of filamentous fungi to chitosan. Molecular microbiology. 2010;75(4):1021-32.) analizó 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. 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 incrementen la fluidez de la membrana plasmática para hacer el hongo más sensible a fungicidas, 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 (5252. 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. 1991;117(2):165-9.). Además, activa algunos procesos de defensa en los tejidos hospederos (5353. 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. 1992;96(9):769-79.), actúa inhibiendo varias enzimas; la unión de la quitosana con el ADN y la inhibición de la síntesis de ARN_m y síntesis de proteínas (5454. Sudarshan NR, Hoover DG, Knorr D. Antibacterial action of chitosan. Food Biotechnology. 1992;6(3):257-72.).

En el caso de las nanopartículas de plata (Ag), se basa en la posibilidad de que estas se adhieran y penetren en la membrana celular causando desbalance osmótico en las esporas, siendo muy efectivas contra Magnaporthe grisea (5555. Jo Y-K, Kim BH, Jung G. Antifungal activity of silver ions and nanoparticles on phytopathogenic fungi. Plant disease. 2009;93(10):1037-43.).

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 linked 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. Prashanth KV, Tharanathan RN. Chitin/chitosan: modifications and their unlimited application potential-an overview. Trends in Food Science & Technology. 2007;18(3):117-31. doi:10.1016/j.tifs.2006.10.022 ). Both the content and the sequence of these units will determine the physicochemical and biological properties of this polymer. Chitosan has a high nitrogen (N) content 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. Chitin. 1977;5-17.). This biopolymer has 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, degree of acetylation, host cell, presence of natural nutrients, chemical or nutritional composition of the substrates and environmental conditions.

Pyricularia oryzae, causal agent of rice blast or rice scorch

Pyricularia and Magnaporthe are currently widely used generic names for the fungus that causes rice scorch. The Pyricularia/ Magnaporthe Working Group has considered the possibility of retaining the name Magnaporthe over Pyricularia. However, such retention would require a change in the type species of the genus Magnaporthe and would cause numerous name changes for those species currently placed in Pyricularia.

The asexually typed generic name, Pyricularia, is the correct name for the rice scorch fungus, which corresponds well with pathogenicity and ecological and evolutionary characteristics. Therefore, the name Pyricularia oryzae should be used for the fungus that produces this disease. However, the synonym Magnaporthe oryzae can continue to be referred to in publications as Pyricularia oryzae (syn. Magnaporthe oryzae). This practice will help to close a potential gap in the literature and knowledge of this important species (1010. Zhang N, Luo J, Rossman AY, Aoki T, Chuma I, Crous PW, et al. Generic names in Magnaporthales. IMA fungus. 2016;7(1):155-9.). As for grisea and oryzae, they are very different species. Grisea is for strains of Digitaria, and oryzae is for strains of rice, wheat and other grasses; although in the literature, they are considered synonyms and all four names are used: Pyricularia oryzae, Magnaporthe oryzae, Pyricularia grisea and Magnaporthe grisea.

Pyricularia oryzae, known as pyriculariosis, blast, blight, blight, leaf blight or rice blight, is one of the most serious diseases of rice (Oryza sativa L.), which has caused significant losses in yields worldwide (1111. 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. 2008;21(2):51-8.). It is a very effective plant pathogen, since it can reproduce sexually (teleomorph: Magnaporthe grisea Barr (It. Hebert) syn. Magnaporthe oryzae) and asexually (anamorph: Pyricularia oryzae). The distribution of the blast is worldwide, as 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 was later found in China (1637), Japan (1760), the United States (1960) and India (1913) (1212. Rossman AY, Howard RJ, Valent B. Pyricularia grisea the correct name for the rice blast disease fungus. Mycologia. 1990;82(4):509-12.). Piriculariosis has caused significant losses, for example: in India 90 % (1313. 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. 2019;8(3):3749-53.), in China 70 % (1414. Ou SH. Rice Diseases: Commonwealth Mycological Institute. 2nd ed. Kew Surrey, England; 1985. 380 p.), 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 (1515. 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. 2009;33(5):487-94.). Mexico has reported a drop in production of up to 30 % in rainfed crops. Cuba has been another country affected by this pathogen, and when conditions are favorable, losses have increased up to 70 %.

Taxonomic classification, morphology and symptomatology of Pyricularia oryzae

The causal agent of pyriculariosis is classified taxonomically in the class: Deuteromycetes, order: Moniliales, family: Dematiaceae, genus: Pyricularia, species: Pyricularia oryzae. Pyricularia oryzae has simple, tabulate, brownish conidiophores (Figure 1 A). The conidiophores are borne singly or in groups of three and carry conidia at their ends. These are hyaline, fusiform and are divided equidistantly by two septa. Their approximate size is 22 to 24 µm x 10 to 12 µm. More than one conidium can form on the conidiophore, the number of conidia ranges from 1 to 20.

It is a complex disease, due to the pathogenic variability and the speed with which this fungus overcomes the resistance of the rice plant. The mycelium of the fungus produces a toxic substance known as pyricularin, which inhibits the growth of tissues and disorganizes them. It attacks the aerial parts of the plant such as leaves, stems, nodes and spikes (1616. 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. 2017;6(6):1154-60.) (Figure 1, B, and C). The fungus produces spots or lesions on the leaves of elongated or elliptical to rhomboid shape, of uniform brown color, which 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 susceptibility of varieties (1717. 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. 2005;26(4):83-7.). 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 the fungus and produces large quantities of spores. The spores arrive from crop residues from the previous season or from weeds, where the fungus has been harbored during the winter (1818. 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. 2010;31(1):14-8.).

General strategies for control of Pyricularia oryzae

Disease control is mainly based on the application of systemic chemical fungicides, including benzimidazoles 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 action mode (1919. Patiño L. La resistencia a fungicidas, una continúa amenaza al control de la Sigatoka Negra. Boletín Técnico Cenibanano. 2003;4:2-5.). 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 the negative effects of conventional fungicides on the environment, questioning their commercial use, being 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 Pyricularia oryzae

The antimicrobial property of chitosan and its derivatives has 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, Penicillum spp, Fusarium oxysporum and Bipolaris oryzae, among other fungi (2020. 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. 2019;246:700-9.-2424. Rodríguez Pedroso AT, Plascencia Jatomea M, Bautista Baños S, Cortez Rocha MO, Ramírez Arrebato MÁ. Actividad antifúngica in vitro de quitosanos sobre patógeno del arroz. Acta Agronomica. 2016;65(2):169-74.).

In this regard, the antifungal activity of chitosan 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 (2121. 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. 2011;99(3):250-5.,2525. Živković S, Stevanović M, Đurović S, Ristić D, Stošić S. Antifungal activity of chitosan against Alternaria alternata and Colletotrichum gloeosporioides. Pesticidi i fitomedicina. 2018;33(3-4):197-204.,2626. 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. 2011;2011.). Some authors have demonstrated the fungicidal effect of these compounds at different concentrations and isolates of 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. 2003;24(2):85-8.,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. 2004;25(4):89-93.,2727. 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. 2005;61(10):951-60.). Also, chitosan oligomers have shown to have a better inhibitory effect on this pathogen, achieving the minimum inhibitory concentration at a concentration higher than 2000 mg L^-1 (2828. 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. 2003;24(2):85-8.). 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. 2004;25(4):89-93.).

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 (2929. 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. 1998;61(9):1124-8.), N,N,N. trimethyl chitosan (3030. Jia Z, Xu W. Synthesis and antibacterial activities of quaternary ammonium salt of chitosan. Carbohydrate research. 2001;333(1):1-6.), N,O-acyl chitosan (3131. Sashiwa H, Kawasaki N, Nakayama A, Muraki E, Yamamoto N, Zhu H, et al. Chemical modification of chitosan. 13. Synthesis of organosoluble, palladium adsorbable, and biodegradable chitosan derivatives toward the chemical plating on plastics. Biomacromolecules. 2002;3(5):1120-5.), O-acyl chitosan (3232. 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.,3333. 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. 2005;70(3):215-8.), hydroxyethyl aryl chitosan (3434. Ma G, Yang D, Tan H, Wu Q, Nie J. Preparation and characterization of N‐alkylated chitosan derivatives. Journal of applied polymer science. 2008;109(2):1093-8.), dimethylpiperazine chitosan (3535. Másson M, Holappa J, Hjálmarsdóttir M, Rúnarsson ÖV, Nevalainen T, Järvinen T. Antimicrobial activity of piperazine derivatives of chitosan. Carbohydrate polymers. 2008;74(3):566-71.), carboxymethyl chitosan (3636. 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. 2008;353(1-2):139-48.), acyl urea thiourea chitosan (3737. 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. 2008;343(3):566-70.), N-succinoyl chitosan (3838. 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 (3939. Stössel P, Leuba JL. Effect of chitosan, chitin and some aminosugars on growth of various soilborne phytopathogenic fungi. Journal of Phytopathology. 1984;111(1):82-90.). Researchers (4040. Badawy ME, Rabea EI. Characterization and antimicrobial activity of water-soluble N-(4-carboxybutyroyl) chitosans against some plant pathogenic bacteria and fungi. Carbohydrate polymers. 2012;87(1):250-6.) have noted that N-alkylation or N-arylation of chitosan with aromatic or aliphatic aldehydes effectively increased its antifungal activity on P. grisea.

With the same methods and obtaining techniques, but using different types of aldehydes, scientists (2929. 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. 1998;61(9):1124-8.) 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; N-(m-nitrobenzyl) chitosan had the greatest effect at a concentration of 5 g L^-1. Also, these authors observed that the most active derivative was N-(2,2 diphenylethyl) chitosan, with a minimum inhibitory concentration of 0.3 g L^-1, against this pathogen. Moreover, 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 (4141. 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. 2008;69(4):973-81.) and momilactones B upon infection with P. grisea in rice leaves at a concentration of 10 µg mL^-1.

A group of researchers published the effect of chitosan on the stimulation of defense responses in rice leaves (4242. 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 the rice leaf. However, treating rice seedlings with 5 mg L^-1 and inoculating them with Magnaporthe grisea 97-23-2D1 showed a better effect and control of the disease by more than 50 % (4343. 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. 2005;162(8):937-44.). 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. 2007;89(3):206-15.). Eighteen days after seed germination, plants obtained were inoculated with P. grisea spores, and the activity of enzymes related to defense, such as PAL, glucanase, chitinase and chitosanase, was evaluated, observing an increase in the activity of the plants treated with the elicitors, with respect to the control. In addition, no disease symptoms were observed in the highest concentration of both compounds used.

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. 2016;84:58-61.,4444. 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.,4545. 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. 2018;18(8):5299-305.). On the other hand, researchers (4444. 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.) found that chitosan and silver (Ag) nanoparticles had a high antifungal activity on Pyricularia oryzae, at a concentration of Ag (2 ppm) and chitosan (4000 ppm). However, other scientists (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. 2016;84:58-61.) applied 500 μl of a 0.1 % chitosan nanoparticle solution on rice leaves, and 24 h later, a spore suspension (1x10⁵ spore mL^-1) of P. grisea, and after 10 days, no disease symptoms were observed.

Mechanisms of chitosan action

Mechanisms of chitosan action of 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 (4646. Je J-Y, Kim S-K. Antimicrobial action of novel chitin derivative. Biochimica et Biophysica Acta (BBA)-General Subjects. 2006;1760(1):104-9.,4747. 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 pH 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 (4848. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM. Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryotic Cell. 2005;4(4):703-15.). 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 (2929. 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. 1998;61(9):1124-8.,4949. 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, or leading to the outgassing of proteins and other intracellular constituents (2929. 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. 1998;61(9):1124-8.). The most abundant of the sphingolipids is mannosyldiinositrophosphate-ceramide (M(IP₂)C), which has two negative charges. Sites where chitosan could bind using its amino groups of glucosamine residues, which are able to interact with the negative components of (M(IP₂)C of the plasma membrane. 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 (4848. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM. Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryotic Cell. 2005;4(4):703-15.). In turn, a working group (5050. 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. 2009;46(8):585-94.,5151. Palma‐Guerrero J, Lopez‐Jimenez JA, Pérez‐Berná AJ, Huang I-C, Jansson H-B, Salinas J, et al. Membrane fluidity determines sensitivity of filamentous fungi to chitosan. Molecular microbiology. 2010;75(4):1021-32.) analyzed the action mode of chitosan 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 showed that different cell types (conidium, germ tube and hyphae) exhibit different sensitivity to chitosan. 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 fluidity of the plasma membrane to make the fungus more sensitive to fungicides, such as chitosan.

Chitosan also acts as a chelating agent that selectively binds trace metals and thereby inhibits toxin production and mycelial growth (5252. 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. 1991;117(2):165-9.). In addition, it activates some defense processes in host tissues (5353. 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. 1992;96(9):769-79.), acts by inhibiting several enzymes; the binding of chitosan to DNA and the inhibition of mRNA synthesis and protein synthesis (5454. Sudarshan NR, Hoover DG, Knorr D. Antibacterial action of chitosan. Food Biotechnology. 1992;6(3):257-72.).

In the case of silver nanoparticles (Ag), it is based on the possibility that these adhere and penetrate the cell membrane causing osmotic imbalance in the spores, being very effective against Magnaporthe grisea (5555. Jo Y-K, Kim BH, Jung G. Antifungal activity of silver ions and nanoparticles on phytopathogenic fungi. Plant disease. 2009;93(10):1037-43.).