Application of energy‐filtering electron microscopy (EFEM) for analysis of hydrogen peroxide and lignin in epidermal walls of cucumber leaves triggered by acibenzolar‐S‐methyl treatment prior to inoculation with Colletotrichum orbiculare

Hydrogen peroxide (H2O2) and lignin induced by acibenzolar‐S‐methyl (ASM) during the expression of systemic acquired resistance (SAR) of cucumber plants against the fungus Colletotrichum orbiculare was analysed with electron microscopy and energy‐filtering electron microscopy (EFEM). ASM pretreatment consistently led to the blockage of appressorial penetration pegs into leaves at early stages after inoculation, whereas the fungus was able to grow into the vascular tissue after distilled water pretreatment. The blocked pegs, seen frequently at epidermal pectin layers in leaves of ASM‐treated plants, were surrounded by abundant electron‐dense amorphous material and dots. These were identified as lignin, based on their appearance and high reactivity with KMnO4. Lignin first appeared as an amorphous material at pectin layers, and then as dots in some areas of the material. The results showed that ASM caused SAR in leaves via faster formation of lignin within 1 day after inoculation. After CeCl3 treatment to detect H2O2, H2O2‐reactive products (cerium perhydroxides) were seen near ASM‐induced lignin formation sites in pectin layers. EFEM analysis showed that Ce and O were located at the same sites as the lignin dots, suggesting that lignin was associated with sources of H2O2 generation. The results indicate that the faster H2O2 generation and lignification induced by ASM were the potential causes of SAR. [ABSTRACT FROM AUTHOR]