Lesion delimitation and resistance of old bean (Phaselous vulgaris L., cv. Red Kidney) plants to Rhizoctonia solani Kuhn 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(.8-aminoethyl ether)-N,NA,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.The subcellular localization of Ca has been the subject of many investigations of animal tissues (7)(8)(9)22). By several procedures, primarily those using fixation with PPA2 or ammonium oxalate, Ca has been reported to occur within mitochondria, Golgi vesicles, sarcoplasmic reticulum, as well as on glycogen particles, heterochromatin, and the nucleolus. In some cases, the presumed localization of Ca was correlated with chemical and electron microprobe analysis of the sections.Fewer studies have concerned the ultrastructural localization of Ca in plant tissues (6, 15, 17,18,20,21). Antimonate precipitates containing Ca were found in the nucleus, mitochondria, dictyosomes, cell wall, phragmoplast, plasma membrane, and ER in cells of both Marsilea sporocarps and Hordeum leaves in a study by Wick and Hepler (21). Antimonate deposits have also been found within nuclei of Allium cepa root and anther cells (18) In a previous study of bean stem canker, cell walls within lesions were severely degraded, whereas walls at the periphery of mature lesions were intact (13). At the lesion margin, walls stained densely following either potassium permanganate or osmium tetroxide fixation. The purpose of the present study was to evaluate methods for Ca localization at the ultrastructural level, to determine whether these methods could be applied to studies of stem canker on beans, and to evaluate the nature of wall changes in older bean plants and in tissue surrounding lesions.
MATERIALS AND METHODS
