Model e 1010

The leaf samples used for observing the morphology of bulliform cell were collected from the youngest fully expanded leaves under control conditions. The sections were prepared with a freezing microtome, and then the tissue slices of the bulliform cells were photographed with a fluorescence microscope (DM4000; Leica, Jena, Germany). The area of the bulliform cells was measured using Image J software (National Institutes of Health, Bethesda, MD, USA) by selecting the calculation tool for irregular delineation.
The number of stomata, their opening rate, and their aperture were determined according to the method of Feng et al. (2018 (link)), that the youngest fully expanded leaves of the rice plants were detached and immediately immersed in 3.5% glutaraldehyde in phosphate buffer (0.1 M, pH = 7.0) at 4 °C. These samples were dehydrated in a Hitachi Model HCP-2 critical point dryer, coated with gold-palladium in the Hitachi Model E-1010 ion sputter for 4–5 min, and observed with a Hitachi Model SU-8010 scanning electron microscope.

For the first-step treatment called double fixation, the foregut was first fixed with 2.5% glutaraldehyde in a phosphate buffer (0.1 M, pH 7.0) for more than 4 h; then, it was washed three times in the phosphate buffer (0.1 M, pH 7.0) for 15 min at each step; then, it was post-fixed with 1% OsO4 in a phosphate buffer for 2 h and washed three times in a phosphate buffer (0.1 M, pH 7.0) for 15 min at each step. Then, the second step was a two-stage dehydration. In the first stage, it was dehydrated by a graded series of ethanol (30%, 50%, 70%, 80%, 90%, and 95%) for about 15 min at each step; then, it was dehydrated two times by alcohol for 20 min at each step, or stored in alcohol. In the second stage, the sample was dehydrated in a Hitachi Model HCP-2 critical point dryer. On the third and last step, the sample was coated with gold-palladium in a Hitachi Model E-1010 ion sputter for 5 min and observed in the Hitachi Model SU-8010 SEM. The image obtained here is a 3D image.

A dry sample was mounted on an aluminum holder and an ion sputtering device (Model E-1010, Hitachi; Tokyo, Japan) was used for gold sputter coating to make it be conductive. The morphology of the samples was characterized by a scanning electron microscope (SU8020, Hitachi; Tokyo, Japan) operating at an accelerating voltage of 5 kV.

The dehydrated samples were coated with gold–palladium in Hitachi Model E-1010 ion sputter for 4–5 min and observed in Hitachi Model SU-8010 SEM.

The microscopic features of the isolated Aspergilli were sequentially studied using Light Microscopy (Olympus CH20i), Scanning Electron (SE; Hitachi, Model E-1010) and Differential Interference Contrast (DIC) microscopic analysis. Prominence was given to study characteristics like hyphal nature, septa, conidiophores, vesicle shape, sterigmata arrangement, and conidia formation. Standard protocols were followed while studying the microscopic features as outlined in identification Keys and Manuals (Thom and Raper, 1945 ; Varga and Samson, 2008 ). The light and scanning electron micrographs were taken at the Fungal Genetics and Mycotoxicology Laboratory, Department of Microbiology, Pondicherry University, while the DIC microscopic images (Nikon Upright Motorised Microscope, ECLIPSE Ni series, Nikon Corporation, Tokyo) were taken at the Fungal Biotechnology Laboratory, Department of Biotechnology, School of Life Sciences, Pondicherry University.

CK and KO-1 were used for observation of outer glume epidermal cell. Young spikelet hulls were fixed with 2.5% glutaraldehyde for 24 h and then dehydrated in a graded series of ethanol (30%, 50%, 75%, 95%, 100%, 100% and 100%). The dehydrated sample were coated with gold–palladium using ion sputter (Model E-1010, Hitachi, Japan) and observed using scanning electron microscope (Model TM-1000, Hitachi, Japan). Cell length and width of the outer glumes were measured, then cell number in the transverse and longitudinal direction were calculated. Twenty glumes were used for CK and KO-1 respectively. The Student’s t-test was employed to test the differences in cell length, width and numbers between CK and KO-1 (p < 0.05).