The fungal hyphae in the infected leaf tissues were harvested and cleared in the Carnot fixator overnight at room temperature. The samples were then rinsed and stained with WGA-Alexa Fluor 488 (Invitrogen, CA, USA), and the plant membrane was visualized using propidium iodide (PI), as described in an earlier study (Gao et al., 2013 (link)). The samples were then observed under a confocal microscope (Zeiss, LSM 780, Germany).
Scanning electron microscopy (SEM) was carried out to observe the infection dynamics of samples collected at different time points after inoculation. The samples were fixed in glutaraldehyde for 2 h and continuously dehydrated with alcohol and tert-butanol, followed by freeze-drying (Vacuum Device, VFD-30, Japan). These samples were coated with gold powder by spraying (Hitachi, MSP-2S, Japan) and observed using an SEM (Hitachi, TM-3030, Japan).
Furthermore, the leaf samples were analyzed by transmission electron microscopy (TEM) to determine the subcellular structural differences. The samples were fixed overnight in glutaraldehyde and dehydrated in acetone. These light microscopy and electron microscopy samples were processed as described by Redkar et al. (2015 (link)). Ultrathin sections were made using a Leica ultramicrotome (Leica, EM UC7, Germany) and observed under a Hitachi TEM (Hitachi, HT7700, Japan).
Scanning electron microscopy (SEM) was carried out to observe the infection dynamics of samples collected at different time points after inoculation. The samples were fixed in glutaraldehyde for 2 h and continuously dehydrated with alcohol and tert-butanol, followed by freeze-drying (Vacuum Device, VFD-30, Japan). These samples were coated with gold powder by spraying (Hitachi, MSP-2S, Japan) and observed using an SEM (Hitachi, TM-3030, Japan).
Furthermore, the leaf samples were analyzed by transmission electron microscopy (TEM) to determine the subcellular structural differences. The samples were fixed overnight in glutaraldehyde and dehydrated in acetone. These light microscopy and electron microscopy samples were processed as described by Redkar et al. (2015 (link)). Ultrathin sections were made using a Leica ultramicrotome (Leica, EM UC7, Germany) and observed under a Hitachi TEM (Hitachi, HT7700, Japan).
Free full text: Click here
