Cytohistochemical techniques for calcium localization and their application to diseased plants

Lesion delimitation and resistance of old bean (Phaselous vulgaris L., cv. Red Kidney) plants to Rhizoctonia solani Kühn have been suggested to result from increased calcium pectate formation in walls. Ultrastructural histochemistry was used to determine the site of calcium in tissues adjacent to lesions and in older bean hypocotyls. Hypocotyl lesion tissue and uninoculated control tissue were treated with ammonium oxalate or potassium pyroantimonate during fixation. Treatment with potassium pyroantimonate, but not with oxalate, resulted in granular deposits in cell walls of healthy and lesion tissue. Granules also occurred on the plasma membrane of cells adjacent to lesions and in organelles of damaged cells, but wall granule density was not increased. Cell walls from healthy 24-day-old plants had a greater granule density than those for 8-day-old plants. Wall granules were removed from thin sections with ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. Energy dispersive analysis of x-rays also suggested that potassium pyroantimonate localized calcium. Chemical analyses showed that some calcium was retained in tissues after fixation. The results suggest that there are different mechanisms for lesion delimitation and age-induced resistance.