Introducción

Los hongos micorrizógenos arbusculares (HMA) (Glomeromycota) son parte integral de numerosos ecosistemas y se consideran particularmente ventajosos, debido a que se asocian a la mayoría de las plantas vasculares estudiadas (11. Berruti A, Lumini E, Balestrini R, Bianciotto V. Arbuscular mycorrhizal fungi as natural biofertilizers: let’s benefit from past successes. Frontiers in microbiology [Internet]. 2016;6:1559. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2015.01559/full ,22. Giovannetti M, Avio L, Sbrana C. Functional significance of anastomosis in arbuscular mycorrhizal networks. In: Mycorrhizal networks [Internet]. Springer; 2015. p. 41-67. Available from: https://link.springer.com/chapter/10.1007/978-94-017-7395-9_2 ). Dichos hongos son biótrofos obligados y colonizan las raíces de sus plantas hospedantes obteniendo azúcares. En cambio, las plantas hospedantes reciben los nutrientes minerales y el agua, absorbidos y transportados a través de una red fina de hifas extrarradicales extendidas, desde las raíces hasta el suelo circundante (33. Bonfante P, Genre A. Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis. Nature communications [Internet]. 2010;1(1):1-11. Available from: https://www.nature.com/articles/ncomms1046?fbclid=IwAR1g7_DQ5BfH6DKqDAuMLO0CIqwsSMMgkEUVRMWED7EVdhpQcloMbKDEMfs ).

La utilización de los HMA resulta factible para cualquier sistema de producción agrícola, debido a las funciones que realizan una vez que se asocian con las plantas. Entre ellas se encuentra el incremento de la absorción de nutrientes minerales y agua, a partir de un aumento en el volumen de suelo explorado; confieren mayor resistencia de las plantas a las toxinas y al ataque de patógenos y el incremento de la translocación y solubilización de elementos esenciales (22. Giovannetti M, Avio L, Sbrana C. Functional significance of anastomosis in arbuscular mycorrhizal networks. In: Mycorrhizal networks [Internet]. Springer; 2015. p. 41-67. Available from: https://link.springer.com/chapter/10.1007/978-94-017-7395-9_2 ,44. Smith SE, Smith FA. Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales. Annual review of plant biology [Internet]. 2011;62:227-50. Available from: https://www.annualreviews.org/doi/abs/10.1146/annurev-arplant-042110-103846 ). Los HMA contribuyen a disminuir los efectos adversos del estrés abiótico, como la presencia de metales pesados y la salinidad de los suelos, así como del estrés biótico, causado por numerosos patógenos (55. Williams A, Manoharan L, Rosenstock NP, Olsson PA, Hedlund K. Long‐term agricultural fertilization alters arbuscular mycorrhizal fungal community composition and barley (H ordeum vulgare) mycorrhizal carbon and phosphorus exchange. New Phytologist [Internet]. 2017;213(2):874-85. Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14196 ,66. Avio L, Turrini A, Giovannetti M, Sbrana C. Designing the ideotype mycorrhizal symbionts for the production of healthy food. Frontiers in plant science [Internet]. 2018;9:1089. Available from: https://www.frontiersin.org/articles/10.3389/fpls.2018.01089/full ).

Por otra parte, en los sistemas de cultivo actuales, se utilizan fungicidas para controlar o eliminar los fitopatógenos fúngicos. Sin embargo, estos productos también podrían afectar la sobrevivencia de microorganismos autóctonos, incluidos aquellos que son beneficiosos para el crecimiento de las plantas, como los HMA. Por lo tanto, el efecto esperado de la micorrización en el crecimiento y el desarrollo de las plantas hospedantes, puede afectarse negativamente por el uso de fungicidas, en los sistemas agrícolas (77. Rivera-Becerril F, van Tuinen D, Chatagnier O, Rouard N, Béguet J, Kuszala C, et al. Impact of a pesticide cocktail (fenhexamid, folpel, deltamethrin) on the abundance of Glomeromycota in two agricultural soils. Science of the Total Environment [Internet]. 2017;577:84-93. Available from: https://www.sciencedirect.com/science/article/abs/pii/S004896971632280X ).

Los fungicidas sistémicos son aquellos que, al ser aplicados al suelo, pueden ser absorbidos por las raíces y transferidos a otras partes de la planta. Son sustancias persistentes y su acción sobre los hongos micorrízicos puede influir negativamente en el desarrollo de sus estructuras vegetativas y reproductivas, por estar diseñados para destruir las quitinas fúngicas, así como otras proteínas y enzimas específicas producidas por estos hongos (88. Battini F, Cristani C, Giovannetti M, Agnolucci M. Multifunctionality and diversity of culturable bacterial communities strictly associated with spores of the plant beneficial symbiont Rhizophagus intraradices. Microbiological research [Internet]. 2016;183:68-79. Available from: https://www.sciencedirect.com/science/article/pii/S0944501315300343 ). Esto inhibe o retrasa la simbiosis entre estos hongos y las plantas, disminuyendo los efectos positivos que los HMA le reportan a la planta, como la absorción de fósforo (88. Battini F, Cristani C, Giovannetti M, Agnolucci M. Multifunctionality and diversity of culturable bacterial communities strictly associated with spores of the plant beneficial symbiont Rhizophagus intraradices. Microbiological research [Internet]. 2016;183:68-79. Available from: https://www.sciencedirect.com/science/article/pii/S0944501315300343 ). En Cuba no existen evidencias documentadas que muestren el efecto de fungicidas sistémicos en la simbiosis HMA-planta.

Teniendo en cuenta los aspectos anteriormente planteados el objetivo del presente estudio fue evaluar el efecto de un fungicida sistémico comercial en la colonización de HMA inoculados en el cultivo del maíz, en condiciones semicontroladas.

Materiales y métodos

Los experimentos se realizaron en el Instituto Nacional de Ciencias Agrícolas (INCA). Se emplearon semillas de maíz cultivar “Raúl Hernández”, obtenido en el Departamento de Genética y Mejoramiento Vegetal del INCA. El experimento se desarrolló en macetas de 5 kg de capacidad, con un sustrato de suelo Pardo Mullido no estéril. El suelo presentó una concentración de 3-5 esporas de HMA residentes por g^-1 de suelo. Algunas características químicas del sustrato empleado se muestran en la Tabla 1.

Se sembraron cinco semillas de maíz por maceta y se inocularon mediante la técnica de recubrimiento de la semilla (99. Fernández F, Rivera R, Noval B. Metodología de recubrimiento de semillas con inoculo micorrizógeno. Patente Cubana. 1999;(22641) .). Se emplearon las cepas de HMA Rhizophagus irregularis (Blaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler (INCAM 11) y Glomus cubense Y. Rodr. & Dalpé (INCAM 4) (registrada en el Herbario Nacional Micológico de Canadá, Ottawa, con código DAOM 241198). Ambas cepas fúngicas pertenecen a la colección de hongos micorrízicos arbusculares del Instituto Nacional de Ciencias Agrícolas de Cuba. Además, se empleó un conglomerado de diferentes esporas de hongos residentes (RPR), aislados de la finca “La Fidelia”, perteneciente a la CCS Oscar Núñez Gil del Municipio Los Palacios. Los aislados se obtuvieron de un suelo Pardo mullido Carbonatado de Los Palacios que también forma parte del cepario del INCA (1010. Hernández-Jiménez A, Pérez-Jiménez JM, Bosch-Infante D, Speck NC. La clasificación de suelos de Cuba: énfasis en la versión de 2015. Cultivos Tropicales [Internet]. 2019;40(1). Available from: http://scielo.sld.cu/scielo.php?pid=S0258-59362019000100015&script=sci_arttext&tlng=pt ). Los inóculos anteriormente mencionados poseían una concentración de 25 esporas g^-1 de suelo fresco.

Se empleó además el fungicida sistémico comercial Previcur energy 840sl (F), el cual está compuesto por un 16 % de ingredientes inertes y como componentes activos presenta un 53 % de propil 3-(dimetilamino) propil carbamato y un 31 % de etilhidrógeno fosfonato. El experimento se condujo utilizando un diseño completamente aleatorizado con arreglo bifactorial. Los factores estudiados fueron las cepas de HMA (factor 1) y la presencia o no del fungicida (factor 2) y se establecieron los siguientes tratamientos, cada uno con siete réplicas (Tabla 2).

A los cinco días de la emergencia de las plántulas, se realizó un raleo y se mantuvo una plántula por maceta. Las plántulas se mantuvieron en condiciones semicontroladas de cultivo por 54 días, a 30 ℃, humedad relativa del 80 % y fotoperiodo natural. A los 15 días de la emergencia de las plantas, se aplicó el fungicida sistémico comercial Previcur energy 840sl, a razón de 10 mL por maceta que presentaba una concentración del fungicida de 10 mg L^-1.

A los 54 días de establecido el experimento, se determinó la densidad visual y la frecuencia de colonización, según lo descrito previamente en la literatura (1111. Trouvelot A, Kough JL, Gianinazzi-Pearson V. Mesure du taux de mycorhization VA d’un système radiculaire. Recherche de méthode d’estimation ayant une signification fonctionnelle. In: Physiological and genetical aspects of mycorrhizae: proceedings of the 1st european symposium on mycorrhizae, Dijon, 1-5 July 1985 [Internet]. 1986. p. 217-21. Available from: https://pascalfrancis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8758731 ). Se determinaron variables que caracterizan el crecimiento de las plantas como la longitud de la raíz, la masa fresca total y la masa seca total de la planta. Para determinar la longitud radical, se empleó una regla graduada y se midió desde el ápice de la raíz hasta el inicio del tallo. La masa fresca total de las plantas se determinó en una balanza técnica (Sartorius), después de lavar y secar con papel de filtro las raíces y la parte aérea. Para determinar la masa seca total, las muestras se secaron en estufa a 75 ℃, durante tres días hasta obtener peso constante.

Los datos se analizaron a partir de un Análisis de varianza de clasificación doble. En el caso en que se encontraran diferencias significativas en los factores evaluados, se realizó un desdoblamiento de la interacción, fijándose uno de los factores, cepa (factor 1) o fungicida (factor 2). Para ello, se empleó una prueba de comparación de medias. Se utilizó el programa STATISTIC versión 21 para el procesamiento estadístico de los datos y el programa Microsoft Excel 2010 para su representación.

Resultados

Como puede apreciarse en la Figura 1 A y B, las variables de frecuencia de colonización y densidad visual tuvieron un comportamiento diferente con relación a los tratamientos evaluados.

No se encontraron diferencias significativas en la frecuencia de colonización entre las plantas inoculadas con y sin el fungicida y las plantas controles. Tampoco se identificaron diferencias entre las plantas que no se trataron con el fungicida y aquellas que sí. Se exceptúa, el tratamiento con RPR y aquel donde se inoculó la cepa INCAM 11, ambos en presencia del fungicida, entre los cuales sí se observaron diferencias (Figura 1 A). Al analizar la densidad visual, ocurrió una disminución significativa de sus valores, en los tratamientos inoculados con INCAM 4 e INCAM 11 en presencia del fungicida, respecto a los tratamientos con estas cepas sin fungicida (Figura 1 B). Sin embargo, las plantas inoculadas con RPR en presencia del fungicida, alcanzaron los mayores valores de densidad visual, con relación al resto de los tratamientos.

Por otra parte, cuando se analizó el efecto de la inoculación y el empleo del fungicida se pudo apreciar que, en la mayor parte de los tratamientos, el empleo del fungicida, tanto en las plantas control como en las inoculadas, incrementó la longitud radical, la masa fresca y seca de las plantas de maíz (Figura 2 y 3 A, B). Sin embargo, entre las plantas inoculadas con la cepa INCAM 11 no se observaron diferencias significativas en ninguna de las variables que se midieron, relacionadas con el crecimiento de las plantas de maíz. Tampoco se observaron diferencias significativas entre los dos grupos de plantas inoculadas con RPR en la masa fresca de las plantas.

Discusión

Se ha informado que el funcionamiento micorrízico y su acción sobre las plantas pueden afectarse por la aplicación de fungicidas, según las condiciones edáficas (1212. Lenoir I, Fontaine J, Sahraoui AL-H. Arbuscular mycorrhizal fungal responses to abiotic stresses: a review. Phytochemistry [Internet]. 2016; 123:4-15. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0031942216300024 ). Tales efectos dependen del ingrediente químico activo de estos productos y de la especie de hongo micorrizógeno (1313. Helander M, Saloniemi I, Omacini M, Druille M, Salminen J-P, Saikkonen K. Glyphosate decreases mycorrhizal colonization and affects plant-soil feedback. Science of the total environment [Internet]. 2018;642:285-91. Available from: https://www.sciencedirect.com/science/article/pii/S0048969718320345 ). El incremento de la longitud de la raíz y masa fresca y seca de las plantas de maíz que se trataron con el fungicida, pueden atribuirse indirectamente a algunos factores. Entre ellos se encuentran el efecto del fungicida en la reducción de los exudados de la raíz y como un agente estresor de la planta huésped. Ambos eventos, favorecen el funcionamiento de la simbiosis micorrízica, con el consiguiente efecto en el crecimiento (1414. Espinosa R, Felix F, Martinez L, Cañizares P, Yakelín R, Ortega E, et al. Manejo, integración y beneficios del biofertilizante micorrízico EcoMic® en la producción agrícola. [Internet]. Ediciones INCA; 2020. Available from: https://www.researchgate.net/publication/340223155_Manejo_integracion_y_beneficios_del_biofertilizante_micorrizico_EcoMicR_en_la_produccion_agricola ,1515. Lekberg Y, Wagner V, Rummel A, McLeod M, Ramsey PW. Strong indirect herbicide effects on mycorrhizal associations through plant community shifts and secondary invasions. Ecological Applications [Internet]. 2017;27(8):2359-68. Available from: https://esajournals.onlinelibrary.wiley.com/doi/abs/10.1002/eap.1613 ).

La diversidad de especies de HMA, presentes en el inoculante de Pinar del Río (RPR), puede ser un factor que explique los elevados valores de densidad visual y de número de esporas, al ser tratadas con el fungicida. En este sentido, se ha demostrado que una mayor diversidad de HMA tiene mayor tolerancia el estrés impuesto por los fungicidas, debido a que algunas especies son más resistentes a ciertos fungicidas que otras (1616. Cameron JC, Lehman RM, Sexton P, Osborne SL, Taheri WI. Fungicidal seed coatings exert minor effects on arbuscular mycorrhizal fungi and plant nutrient content. Agronomy Journal [Internet]. 2017;109(3):1005-12. Available from: https://acsess.onlinelibrary.wiley.com/doi/abs/10.2134/agronj2016.10.0597 ,1717. Brigido C, van Tuinen D, Brito I, Alho L, Goss MJ, Carvalho M. Management of the biological diversity of AM fungi by combination of host plant succession and integrity of extraradical mycelium. Soil Biology and Biochemistry [Internet]. 2017;112:237-47. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0038071717305011 ). Se considera, que la densidad visual refleja con mayor claridad la ocupación del micelio y de hecho, la propia eficiencia simbiótica debido a que expresa la cantidad porcentual de estructuras fúngicas y de intercambio entre los simbiontes. Esto incluye los niveles de colonización y las estructuras de intercambio que se observan durante la evaluación (1818. Schlaeppi K, Bender SF, Mascher F, Russo G, Patrignani A, Camenzind T, et al. High‐resolution community profiling of arbuscular mycorrhizal fungi. New Phytologist [Internet]. 2016;212(3):780-91. Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14070 ). De esta manera, se puede deducir, que las especies presentes en el conglomerado RPR fueran las más eficientes en las condiciones en que se desempeñó el trabajo experimental, al presentar los mayores niveles de densidad visual.

Lo anteriormente planteado, se corresponde con estudios previos, los cuales informaron que la formación de vesículas de HMA en las raíces es significativamente mayor cuando se aplicaron fungicidas como Captan y Tebuconazol, en semillas de maíz (1919. Hage‐Ahmed K, Rosner K, Steinkellner S. Arbuscular mycorrhizal fungi and their response to pesticides. Pest management science [Internet]. 2019;75(3):583-90. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/ps.5220 ). La aplicación del fungicida Metalaxilo incrementó la colonización de HMA de plantas de maíz y pueden estimular también el desarrollo del micelio fúngico, en plantas de Ananas comosus (piña) (1515. Lekberg Y, Wagner V, Rummel A, McLeod M, Ramsey PW. Strong indirect herbicide effects on mycorrhizal associations through plant community shifts and secondary invasions. Ecological Applications [Internet]. 2017;27(8):2359-68. Available from: https://esajournals.onlinelibrary.wiley.com/doi/abs/10.1002/eap.1613 ,1919. Hage‐Ahmed K, Rosner K, Steinkellner S. Arbuscular mycorrhizal fungi and their response to pesticides. Pest management science [Internet]. 2019;75(3):583-90. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/ps.5220 ). Esto puede ocurrir por varias razones, primero una acción directa del fungicida sobre el HMA desencadenando una respuesta positiva, al estimular la producción de arbúsculos (estructura de intercambio del HMA) y también lo constituye la presencia de moléculas xenobióticas en la planta que pudiera inducir algún estrés que aprovecha oportunamente el HMA para expresar mayor colonización y densidad visual.

Otros autores plantean que el dominio de una especie de HMA en un ambiente específico se puede atribuir a diversos factores ambientales. Entre ellos se encuentran las propiedades fisicoquímicas del suelo, la morfología de la planta, la compatibilidad entre planta hospedante y especies de HMA y también la aplicación de los fungicidas (1818. Schlaeppi K, Bender SF, Mascher F, Russo G, Patrignani A, Camenzind T, et al. High‐resolution community profiling of arbuscular mycorrhizal fungi. New Phytologist [Internet]. 2016;212(3):780-91. Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14070 ). Por lo tanto, el empleo del fungicida sistémico, pudiera explicar los bajos valores de densidad visual encontrados en las plantas inoculadas con la cepa INCAM 4 y el fungicida. Estos hallazgos coinciden con algunos estudios, donde se corrobora que los fungicidas sistémicos pueden tener un efecto supresor sobre la actividad de los HMA e incluso en la fisiología de la interacción planta-HMA y en la absorción de nutrientes y agua (1818. Schlaeppi K, Bender SF, Mascher F, Russo G, Patrignani A, Camenzind T, et al. High‐resolution community profiling of arbuscular mycorrhizal fungi. New Phytologist [Internet]. 2016;212(3):780-91. Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14070 ,1919. Hage‐Ahmed K, Rosner K, Steinkellner S. Arbuscular mycorrhizal fungi and their response to pesticides. Pest management science [Internet]. 2019;75(3):583-90. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/ps.5220 ).

Un estudio similar al anterior, se realizó con la aplicación del fungicida Benomyl, el cual provocó un efecto negativo sobre algunos HMA, específicamente sobre el crecimiento del micelio extrarradical, teniendo un efecto directo sobre la longitud del tubo germinativo (2020. Kjøller R, Rosendahl S. Effects of fungicides on arbuscular mycorrhizal fungi: differential responses in alkaline phosphatase activity of external and internal hyphae. Biology and Fertility of Soils [Internet]. 2000;31(5):361-5. Available from: https://link.springer.com/article/10.1007/s003749900180 ). Del mismo modo, los fungicidas Bavistin y Mancozeb tuvieron un efecto negativo en la colonización de las raíces micorrizadas y el número de esporas (2121. O’Connor P, Manjarrez M, Smith SE. The fate and efficacy of benomyl applied to field soils to suppress activity of arbuscular mycorrhizal fungi. Canadian journal of microbiology [Internet]. 2009;55(7):901-4. Available from: https://cdnsciencepub.com/doi/abs/10.1139/W09-035 ).

El Bencimidazol, otro fungicida también muy empleado, ha demostrado ser perjudicial para las micorrizas y para la comunidad microbiológica del suelo. En algunos casos, su uso causa una reducción en el crecimiento de las plantas y la absorción de fósforo (2121. O’Connor P, Manjarrez M, Smith SE. The fate and efficacy of benomyl applied to field soils to suppress activity of arbuscular mycorrhizal fungi. Canadian journal of microbiology [Internet]. 2009;55(7):901-4. Available from: https://cdnsciencepub.com/doi/abs/10.1139/W09-035 ).

Los fungicidas Fenhexamida, Dicamba, Benomilo entre otros, probados en estudios realizados (2121. O’Connor P, Manjarrez M, Smith SE. The fate and efficacy of benomyl applied to field soils to suppress activity of arbuscular mycorrhizal fungi. Canadian journal of microbiology [Internet]. 2009;55(7):901-4. Available from: https://cdnsciencepub.com/doi/abs/10.1139/W09-035 ), redujeron significativamente la densidad y longitud del micelio extrarradical de la cepa de HMA Funneliformis mosseae. De igual modo su viabilidad y la capacidad de explorar la superficie de las membranas de las plantas se vieron negativamente afectadas especialmente por el Fenhexamida.

Conclusiones

Introduction

Arbuscular mycorrhizal fungi (AMF) (Glomeromycota) are an integral part of many ecosystems and they are considered particularly advantageous because they are associated with most of the vascular plants studied (11. Berruti A, Lumini E, Balestrini R, Bianciotto V. Arbuscular mycorrhizal fungi as natural biofertilizers: let’s benefit from past successes. Frontiers in microbiology [Internet]. 2016;6:1559. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2015.01559/full ,22. Giovannetti M, Avio L, Sbrana C. Functional significance of anastomosis in arbuscular mycorrhizal networks. In: Mycorrhizal networks [Internet]. Springer; 2015. p. 41-67. Available from: https://link.springer.com/chapter/10.1007/978-94-017-7395-9_2 ). These fungi are obligate biotrophs and colonize roots of their host plants, obtaining sugars. Instead, host plants receive mineral nutrients and water, absorbed and transported through a fine network of extended extraradical hyphae from roots to the surrounding soil (33. Bonfante P, Genre A. Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis. Nature communications [Internet]. 2010;1(1):1-11. Available from: https://www.nature.com/articles/ncomms1046?fbclid=IwAR1g7_DQ5BfH6DKqDAuMLO0CIqwsSMMgkEUVRMWED7EVdhpQcloMbKDEMfs ).

AMF use is feasible for any agricultural production system because of the functions they perform once associated with plants. Among them is the increase in the absorption of mineral nutrients and water, from an increase in the volume of soil explored; they confer greater plant resistance to toxins and pathogen attack and increase the translocation and solubilization of essential elements (22. Giovannetti M, Avio L, Sbrana C. Functional significance of anastomosis in arbuscular mycorrhizal networks. In: Mycorrhizal networks [Internet]. Springer; 2015. p. 41-67. Available from: https://link.springer.com/chapter/10.1007/978-94-017-7395-9_2 ,44. Smith SE, Smith FA. Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales. Annual review of plant biology [Internet]. 2011;62:227-50. Available from: https://www.annualreviews.org/doi/abs/10.1146/annurev-arplant-042110-103846 ). AMF contribute to reducing the adverse effects of abiotic stress, such as the presence of heavy metals and soil salinity, as well as biotic stress, caused by numerous pathogens (55. Williams A, Manoharan L, Rosenstock NP, Olsson PA, Hedlund K. Long‐term agricultural fertilization alters arbuscular mycorrhizal fungal community composition and barley (H ordeum vulgare) mycorrhizal carbon and phosphorus exchange. New Phytologist [Internet]. 2017;213(2):874-85. Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14196 ,66. Avio L, Turrini A, Giovannetti M, Sbrana C. Designing the ideotype mycorrhizal symbionts for the production of healthy food. Frontiers in plant science [Internet]. 2018;9:1089. Available from: https://www.frontiersin.org/articles/10.3389/fpls.2018.01089/full ).

On the other hand, in current cropping systems, fungicides are used to control or eliminate fungal phytopathogens. However, these products could also affect the survival of indigenous microorganisms, including those that are beneficial to plant growth, such as AMF. Therefore, the expected effect of mycorrhization on host plant growth and development may be negatively affected by the use of fungicides in agricultural systems (77. Rivera-Becerril F, van Tuinen D, Chatagnier O, Rouard N, Béguet J, Kuszala C, et al. Impact of a pesticide cocktail (fenhexamid, folpel, deltamethrin) on the abundance of Glomeromycota in two agricultural soils. Science of the Total Environment [Internet]. 2017;577:84-93. Available from: https://www.sciencedirect.com/science/article/abs/pii/S004896971632280X ).

Systemic fungicides are those that, when applied to the soil, can be absorbed by roots and transferred to other parts of the plant. They are persistent substances and their action on mycorrhizal fungi can negatively influence the development of their vegetative and reproductive structures, because they are designed to destroy fungal chitins, as well as other specific proteins and enzymes produced by these fungi (88. Battini F, Cristani C, Giovannetti M, Agnolucci M. Multifunctionality and diversity of culturable bacterial communities strictly associated with spores of the plant beneficial symbiont Rhizophagus intraradices. Microbiological research [Internet]. 2016;183:68-79. Available from: https://www.sciencedirect.com/science/article/pii/S0944501315300343 ). This inhibits or delays the symbiosis between these fungi and plants, diminishing positive effects that AMF have on the plant, such as phosphorus absorption (88. Battini F, Cristani C, Giovannetti M, Agnolucci M. Multifunctionality and diversity of culturable bacterial communities strictly associated with spores of the plant beneficial symbiont Rhizophagus intraradices. Microbiological research [Internet]. 2016;183:68-79. Available from: https://www.sciencedirect.com/science/article/pii/S0944501315300343 ). In Cuba, there is no documented evidence showing the effect of systemic fungicides on AMF-plant symbiosis.

For this, the aim of the present study was to evaluate the effect of a commercial systemic fungicide on the AMF colonization inoculated on corn crops, under semi-controlled conditions.

Materials and methods

Experiments were conducted at the National Institute of Agricultural Sciences (INCA). Seeds of maize cultivar "Raul Hernandez", obtained from the Department of Genetics and Plant Breeding of INCA, were used. The experiment was carried out in 5 kg pots, with a substrate of non-sterile brown fluffy soil. The soil had a concentration of 3-5 resident AMF spores per g^-1 of soil. Some chemical characteristics of the substrate used are shown in Table 1.

Five maize seeds were sown per pot and inoculated using the seed coating technique (99. Fernández F, Rivera R, Noval B. Metodología de recubrimiento de semillas con inoculo micorrizógeno. Patente Cubana. 1999;(22641) .). Rhizophagus irregularis (Blaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler (INCAM 11) and Glomus cubense Y. Rodr & Dalpé (INCAM 4) AMF strains were used (registered at the National Mycological Herbarium of Canada, Ottawa, with code DAOM 241198). Both fungal strains belong to the collection of arbuscular mycorrhizal fungi of the National Institute of Agricultural Sciences of Cuba. In addition, a cluster of different resident fungal spores (RPR), isolated from "La Fidelia" farm, belonging to the CCS "Oscar Núñez Gil" from Los Palacios municipality, was employed. The isolates were obtained from a soft brown carbonate soil of Los Palacios that is also part of the INCA strain collection (1010. Hernández-Jiménez A, Pérez-Jiménez JM, Bosch-Infante D, Speck NC. La clasificación de suelos de Cuba: énfasis en la versión de 2015. Cultivos Tropicales [Internet]. 2019;40(1). Available from: http://scielo.sld.cu/scielo.php?pid=S0258-59362019000100015&script=sci_arttext&tlng=pt ). The aforementioned inoculums had a concentration of 25 g^-1 spores of fresh soil.

In addition, the commercial systemic fungicide Previcur energy 840sl (F) was used, which is composed of 16 % inert ingredients and as active components has 53 % propyl 3-(dimethylamino) propyl carbamate and 31 % ethylhydrogen phosphonate. The experiment was conducted using a completely randomized design with a bifactorial arrangement. The factors studied were AMF strains (factor 1) and the presence or not of the fungicide (factor 2) and the following treatments were established, each with seven replicates (Table 2).

Five days after seedling emergence, seedlings were thinned and one seedling per pot was maintained. Seedlings were maintained under semi-controlled growing conditions for 54 days, at 30 ºC, 80 % relative humidity and natural photoperiod. Fifteen days after plant emergence, the commercial systemic fungicide Previcur energy 840sl was applied at a rate of 10 mL per pot with a fungicide concentration of 10 mg L^-1.

Fifty-four days after the experiment was established, visual density and colonization frequency were determined, as previously described in the literature (1111. Trouvelot A, Kough JL, Gianinazzi-Pearson V. Mesure du taux de mycorhization VA d’un système radiculaire. Recherche de méthode d’estimation ayant une signification fonctionnelle. In: Physiological and genetical aspects of mycorrhizae: proceedings of the 1st european symposium on mycorrhizae, Dijon, 1-5 July 1985 [Internet]. 1986. p. 217-21. Available from: https://pascalfrancis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8758731 ). Variables characterizing plant growth such as root length, total fresh mass and total dry mass of the plant were determined. To determine root length, a graduated ruler was used and measured from the root apex to the stem begins. The total fresh mass of the plants was determined on a technical balance (Sartorius), after washing and drying roots and aerial part with filter paper. To determine the total dry mass, the samples were dried in an oven at 75 ºC for three days until a constant weight was obtained.

Data were analyzed using a double classification analysis of variance. If significant differences were found in the factors evaluated, the interaction was split, fixing one of the factors, strain (factor 1) or fungicide (factor 2). For this purpose, a mean comparison test was used. The STATISTIC version 21 program was used for the statistical processing of the data and Microsoft Excel 2010 was used for their representation.

Results

As can be seen in Figure 1 A and B, the variables of colonization frequency and visual density behaved differently in relation to treatments evaluated.

No significant differences were found in the frequency of colonization between plants inoculated with and without the fungicide and control plants. Nor were differences identified between plants not treated with the fungicide and those treated with the fungicide. With the exception of the treatment with RPR and that with INCAM 11 strain inoculation, both in the presence of the fungicide, between which differences were observed (Figure 1 A). When visual density was analyzed, there was a significant decrease in its values in the treatments inoculated with INCAM 4 and INCAM 11 in the fungicide presence, with respect to treatments with these strains without fungicide (Figure 1 B). However, the plants inoculated with RPR in the presence of the fungicide reached the highest visual density values compared to the rest of treatments.

On the other hand, when the effect of inoculation and the use of the fungicide was analyzed, it could be seen that, in most of the treatments, the fungicide use, both in control and inoculated plants, increased the root length and the fresh and dry mass of the corn plants (Figure 2 and 3 A, B).

However, among plants inoculated with INCAM 11 strain, no significant differences were observed in any of the variables measured, related to the growth of maize plants. Nor were significant differences observed between the two groups of plants inoculated with RPR in the fresh mass of plants.

Discussion

It has been reported that mycorrhizal functioning and its action on plants can be affected by the application of fungicides, depending on soil conditions (1212. Lenoir I, Fontaine J, Sahraoui AL-H. Arbuscular mycorrhizal fungal responses to abiotic stresses: a review. Phytochemistry [Internet]. 2016; 123:4-15. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0031942216300024 ). Such effects depend on the active chemical ingredient of these products and mycorrhizal fungus species (1313. Helander M, Saloniemi I, Omacini M, Druille M, Salminen J-P, Saikkonen K. Glyphosate decreases mycorrhizal colonization and affects plant-soil feedback. Science of the total environment [Internet]. 2018;642:285-91. Available from: https://www.sciencedirect.com/science/article/pii/S0048969718320345 ). The increase in root length and fresh and dry mass of corn plants treated with the fungicide can be indirectly attributed to some factors. Among them are the fungicide effect in reducing root exudates and as a stressor of the host plant. Both events enhances the mycorrhizal symbiosis functioning, with a consequent effect on growth (1414. Espinosa R, Felix F, Martinez L, Cañizares P, Yakelín R, Ortega E, et al. Manejo, integración y beneficios del biofertilizante micorrízico EcoMic® en la producción agrícola. [Internet]. Ediciones INCA; 2020. Available from: https://www.researchgate.net/publication/340223155_Manejo_integracion_y_beneficios_del_biofertilizante_micorrizico_EcoMicR_en_la_produccion_agricola ,1515. Lekberg Y, Wagner V, Rummel A, McLeod M, Ramsey PW. Strong indirect herbicide effects on mycorrhizal associations through plant community shifts and secondary invasions. Ecological Applications [Internet]. 2017;27(8):2359-68. Available from: https://esajournals.onlinelibrary.wiley.com/doi/abs/10.1002/eap.1613 ).

The diversity of AMF species present in the inoculant from Pinar del Río (RPR) may be a factor that explains high values of visual density and number of spores when treated with the fungicide. In this sense, it has been demonstrated that a greater AMF diversity has better tolerance to the stress imposed by fungicides, due to the fact that some species are more resistant to certain fungicides than others (1616. Cameron JC, Lehman RM, Sexton P, Osborne SL, Taheri WI. Fungicidal seed coatings exert minor effects on arbuscular mycorrhizal fungi and plant nutrient content. Agronomy Journal [Internet]. 2017;109(3):1005-12. Available from: https://acsess.onlinelibrary.wiley.com/doi/abs/10.2134/agronj2016.10.0597 ,1717. Brigido C, van Tuinen D, Brito I, Alho L, Goss MJ, Carvalho M. Management of the biological diversity of AM fungi by combination of host plant succession and integrity of extraradical mycelium. Soil Biology and Biochemistry [Internet]. 2017;112:237-47. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0038071717305011 ).

Visual density is considered to more clearly reflect mycelial occupancy and, indeed, symbiotic efficiency itself because it expresses the percentage amount of fungal structures and exchange between symbionts. This includes levels of colonization and exchange structures observed during evaluation (1818. Schlaeppi K, Bender SF, Mascher F, Russo G, Patrignani A, Camenzind T, et al. High‐resolution community profiling of arbuscular mycorrhizal fungi. New Phytologist [Internet]. 2016;212(3):780-91. Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14070 ). Thus, it can be deduced that species present in the RPR cluster were the most efficient in conditions in which the experimental work was carried out, presenting the highest levels of visual density.

This corresponds to previous studies, which reported that the AMF vesicles formation in the roots is significantly higher when fungicides such as Captan and Tebuconazole were applied to corn seeds (1919. Hage‐Ahmed K, Rosner K, Steinkellner S. Arbuscular mycorrhizal fungi and their response to pesticides. Pest management science [Internet]. 2019;75(3):583-90. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/ps.5220 ). Application of the fungicide Metalaxyl increased AMF colonization of maize plants and may also stimulate fungal mycelium development on Ananas comosus (pineapple) plants (1515. Lekberg Y, Wagner V, Rummel A, McLeod M, Ramsey PW. Strong indirect herbicide effects on mycorrhizal associations through plant community shifts and secondary invasions. Ecological Applications [Internet]. 2017;27(8):2359-68. Available from: https://esajournals.onlinelibrary.wiley.com/doi/abs/10.1002/eap.1613 ,1919. Hage‐Ahmed K, Rosner K, Steinkellner S. Arbuscular mycorrhizal fungi and their response to pesticides. Pest management science [Internet]. 2019;75(3):583-90. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/ps.5220 ). This can occur for several reasons, first a fungicide direct action on AMF triggering a positive response by stimulating the production of arbuscules (AMF exchange structure) and also the presence of xenobiotic molecules in the plant that could induce some stress that takes advantage of AMF to express greater colonization and visual density.

Other authors suggest that the dominance of an AMF species in a specific environment can be attributed to various environmental factors. These include soil physicochemical properties, plant morphology, compatibility between host plant and AMF species, and also fungicide application (1818. Schlaeppi K, Bender SF, Mascher F, Russo G, Patrignani A, Camenzind T, et al. High‐resolution community profiling of arbuscular mycorrhizal fungi. New Phytologist [Internet]. 2016;212(3):780-91. Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14070 ). Therefore, the use of the systemic fungicide could explain the low visual density values found in plants inoculated with INCAM 4 strain and the fungicide. These findings coincide with some studies, which corroborate that systemic fungicides can have a suppressive effect on AMF activity and even on the physiology of plant-AMF interaction and on nutrient and water absorption (1818. Schlaeppi K, Bender SF, Mascher F, Russo G, Patrignani A, Camenzind T, et al. High‐resolution community profiling of arbuscular mycorrhizal fungi. New Phytologist [Internet]. 2016;212(3):780-91. Available from: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14070 ,1919. Hage‐Ahmed K, Rosner K, Steinkellner S. Arbuscular mycorrhizal fungi and their response to pesticides. Pest management science [Internet]. 2019;75(3):583-90. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/ps.5220 ).

A study similar to the previous one was carried out with the application of Benomyl fungicide, which caused a negative effect on some AMF, specifically on the growth of extraradical mycelium, having a direct effect on the germinative tube length (2020. Kjøller R, Rosendahl S. Effects of fungicides on arbuscular mycorrhizal fungi: differential responses in alkaline phosphatase activity of external and internal hyphae. Biology and Fertility of Soils [Internet]. 2000;31(5):361-5. Available from: https://link.springer.com/article/10.1007/s003749900180 ). Similarly, Bavistin and Mancozeb fungicides had a negative effect on mycorrhizal root colonization and spore number (2121. O’Connor P, Manjarrez M, Smith SE. The fate and efficacy of benomyl applied to field soils to suppress activity of arbuscular mycorrhizal fungi. Canadian journal of microbiology [Internet]. 2009;55(7):901-4. Available from: https://cdnsciencepub.com/doi/abs/10.1139/W09-035 ).

Benzimidazole, another widely used fungicide, has been shown to be detrimental to mycorrhizae and the soil microbiological community. In some cases, its use causes a reduction in plant growth and phosphorus uptake (2121. O’Connor P, Manjarrez M, Smith SE. The fate and efficacy of benomyl applied to field soils to suppress activity of arbuscular mycorrhizal fungi. Canadian journal of microbiology [Internet]. 2009;55(7):901-4. Available from: https://cdnsciencepub.com/doi/abs/10.1139/W09-035 ).

Fenhexamide, Dicamba, Benomyl fungicides among others, tested in studies (2121. O’Connor P, Manjarrez M, Smith SE. The fate and efficacy of benomyl applied to field soils to suppress activity of arbuscular mycorrhizal fungi. Canadian journal of microbiology [Internet]. 2009;55(7):901-4. Available from: https://cdnsciencepub.com/doi/abs/10.1139/W09-035 ), significantly reduced the density and the extraradical mycelium length of the Funneliformis mosseae AMF strain. Similarly, their viability and ability to explore the surface of plant membranes were negatively affected, especially by Phenhexamid.

Conclusions