Ultrastructure of mature oospores of Phytophthora megasperma f.sp. medicaginis: Preparation protocols and effects of MTT vital staining and permanganate pre-treatment

Abstract

The ultrastructural cytology of mature oospores of Phytophthora megasperma f.sp. medicaginis is described. Vacuum infiltration with an Epon-based resin gave the best overall preservation, particularly of the thick inner oospore wall, which usually showed considerable shrinkage with routine embedding protocols. The central ooplast vacuole is readily displaced and distorted by centrifugation, which is best avoided during fixation. The cytoplasmic membranes stained with negative contrast. The single oospore nucleus frequently had elaborate loop-like projections and was associated with endoplasmic reticulum and unidentifiable membrane-bound organelles. Other organelles distributed amongst the peripheral lipid contained electron-dense whorled material and were possibly homologous with the larger ooplast vacuole. Cytoplasmic ribosomes were not observed, although vital staining with tetrazolium bromide (MTT) resulted in a fine granular deposit throughout the cytoplasmic matrix. Brief pre-treatment of the oospores with 0.1% potassium permanganate, which dramatically enhanced germination, had no detectable effect on the oospore cytoplasm. It did, however, reveal the microfibrillar substructure of the thick inner layer of the oospore wall. Abnormal, usually non-viable spores were often associated with an irregular, disrupted, outer oospore wall layer.