Cytohistochemical Techniques for Calcium Localization and Their Application to Diseased Plants

Abstract

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

Mechanism of biological control of Rhizoctonia damping-off of cucumber by a non-pathogenic isolate of binucleate Rhizoctonia

“…Studies conducted regarding the presence of calcium cations in resistant host tissues showed its importance in disease control (Bateman and Lumsden, 1965;Stockwell and Hanchey, 1982;Akino et al, 1987;Yang et al, 1993;Biggs et al, 1994). Crystal deposits of calcium salts observed on the hypocotyls surface of BNR-treated seedlings ( Figures 3A and B) proved its essential role in the suppression of damping-off in cucumber.…”

Impact of Zebra Chip Disease on the Mineral Content of Potato Tubers

“…atroseptica both in laboratory and field experiments (McGuire and Kelman 1984;McGuire and Kelman 1986). The increase in tissue calcium levels has been reported to result in a decrease in the activity of pectolytic enzymes resulting in resistance to cell wall degradation (Bateman 1964;Bateman and Lumsden 1965;Stockwell and Hanchey 1982;Weintraub and Ragetli 1961;reviewed in Lambert et al 2005). Magnesium solutions infiltrated into potato tubers also reduced the level of soft rot severity, but not to the same extent as infiltrations made with calcium (McGuire and Kelman 1986).…”

“…The mineral status of potato tuber calcium has also been investigated and shown to be a determining factor in resistance and/or susceptibility to many pathogenic diseases, including reduced severity of bacterial soft rot (Erwinia carotovora) in potato tubers with increased calcium content (McGuire and Kelman 1984;Cother and Cullis 1992;Kleinhenz et al 1995), and common scab (Streptomyces scabies) (Lacey and Wilson 2001). An increase in tissue calcium levels has been reported to result in a decrease in the activity of pectolytic enzymes resulting in resistance to cell wall degradation (Bateman 1964;Bateman and Lumsden 1965;Stockwell and Hanchey 1982;Weintraub and Ragetli 1961;reviewed in Lambert et al 2005). In addition to published data connecting certain minerals to resistance and/or susceptibility to various potato diseases, work has also been conducted looking at the direct effects of potato diseases on the mineral composition of tubers: PVX (Chandra and Mondy 1981), common scab (Krištůfek et al 2000), and leaf roll virus (Hoveland et al 1954;Ulrychová and Limberk 1968).…”

Complementary analysis of freeze-dried and frozen-hydrated plant tissue by electron-probe X-ray microanalysis: Spectral resolution and analysis of calcium

“…Leigh et al 1986;Stockwell and Hanchey 1982). Several reports in the literature, however, have presented EPMA results for Ca under conditions where the presence of K is either demonstrated or could be assumed, and where no attempt has been made to separate the counts of the mixed (Kk~ and Cak,) peak (e.g.…”