Protocolos de aislamiento y diagnóstico de Phytophthora Spp.: enfoque aplicado a la investigación

Abstract

ResumenEl propósito de esta revisión es dar a conocer al investigador, protocolos fundamentales que se emplean para el aislamiento, diagnóstico e identificación de Phytophthora spp., cubriendo aspectos generales del género para una mejor comprensión y análisis del mismo. Esta información será de utilidad para investigadores que trabajan con enfermedades causadas por este oomicete, y va dirigida a países de América Latina. Actualmente, se emplean métodos convencionales y moleculares para la detección e identificación de especies de Phytophthora. Los métodos convencionales incluyen el uso de claves taxonómicas, la observación de síntomas y detección de signos del patógeno en las plantas enfermas y el aislamiento del agente causal del tejido vegetal infectado, del suelo que rodea a las raíces y de las fuentes de agua utilizadas para el riego, usando medios de cultivo generales o selectivos. Los métodos moleculares proporcionan resultados que son más precisos en la identificación de Phytophthora, además de que se pueden realizar en lapsos de tiempo más cortos y no es indispensable que el investigador tenga experiencia en AbstractThe purpose of this review is to make known to the researcher, fundamental protocols that are used for the isolation, diagnosis and identification of Phytophthora spp., covering general aspects of the genus for a better understanding and analysis of the same. This information will be useful for researchers working with diseases caused by this oomycete, and is mainly addressed to countries in Latin America. Currently, conventional and molecular methods are used for the detection and identification of Phytophthora species. Conventional methods include the use of taxonomic keys, observation of symptoms and detection of signs of the pathogen in diseased plants, and isolation of the causative agent of infected plant tissue, soil surrounding roots and water sources used for irrigation, using general or selective culture media. Molecular methods provide results that are more accurate in Phytophthora identification and can be performed in shorter time periods and it is not essential that the researcher has experience in aspects of the morphology of members

Morphological characterization of Phytophthora capsici isolates from Jalisco and Michoacán, Mexico

“…The boxes were sealed with plastic paraffin (Parafilm ® ) and stored at 24 °C for 36-48 h or until the petiole became necrotic. Later on, the petiole was transferred to a NARPH medium with natamycin (0.02 g L -1 ), ampicillin (0.27 g L -1 ), rifampicin (0.01 g L -1 ), pentachloronitrobenzene (0.10 g L -1 ) and hymexazol (0.075 g L -1 ) and stored at 24 °C (Soto-Plancarte et al, 2017).…”

“…Las cajas se sellaron con parafina plástica Parafilm ® y se colocaron a 24 °C durante 36-48 h o hasta la presencia de necrosis en el pecíolo. Posteriormente, el pecíolo se transfirió a medio NARPH con natamicina (0.02 g L -1 ), ampicilina (0.27 g L -1 ), rifampicina (0.01 g L -1 ), pentacloronitrobenceno (0.10 g L -1 ) e himexazol (0.075 g L -1 ) y se almacenó a 24 °C (Soto-Plancarte et al, 2017).…”

“…Los trozos de tejido se desinfestaron en una solución de cloro comercial 10% v/v (0.6% ingrediente activo de hipoclorito de sodio). Posteriormente, se enjuagaron en agua destilada estéril y se colocaron en papel secante estéril de acuerdo al procedimiento descrito por Soto-Plancarte et al (2017). Los trozos de tejido se sembraron en medio NARPH.…”

“…To determine if the isolates had contaminant bacteria, an agar disk containing mycelium was kept in a test tube with sterile Luria Bertani medium at 24 °C for 24 h; the isolate was considered free of bacteria when no turbidity was observed. When bacteria contamination was observed, the culture was transferred to agarpotato-dextrose medium with 0.14% tartaric acid (Soto-Plancarte et al, 2017). Then, medium disks with 5-7 days-old mycelium were transferred to microtubes containing sterile distilled water and kept at 15 °C.…”

“…When the isolates sporulated, the asexual traits were described. The test to determine sporangia expiration was conducted as follows: a drop of water was deposited Plancarte et al, 2017). Posteriormente, se transfirieron discos de medio con micelio de 5-7 días de edad para su conservación en agua destilada estéril dentro de micro-tubos a 15 °C.…”