Sterol Changes in Maize Leaves Infected with Helminthosporium carbonum.

Results of studies with polyene antibiotics have pointed to an interaction with membranes (1). These interactions were reported to occur specifically with sterols (2), and a sparing effect has been noticed upon addition of exogenous sterols (4). It has also been noted that certain phytotoxins produced by plant pathogens interact with plant cell membranes, affecting their permeability and occasionally causing them to rupture (7, 12, 15). Still other work has implicated sterols in membrane structure (14), and the addition of exogenous sterols was found to exhibit a sparing effect against the action of certain chemicals which induce a loss of electrolytes from plant cells (5). Since permeability may be involved in some mechanisms of disease resistance, it appeared logical to study a particular hostpathogen combination which involved a toxin (13) and to determine what differences, if any, existed in sterol content between healthy and inoculated resistant and susceptible host plants. For this purpose maize and Helminthosporium carbonum (Ullstrup) were chosen as the host-pathogen combination. Susceptible hybrids (PrxK61) and resistant hybrids (Prl X K61) of maize are referred to hereafter as Pr and Prl, respectively. The latter is resistant to both races 1 and 2 of this pathogen but Pr is susceptible to race 1 and resistant to race 2. Culture and inoculation conditions are reported fully elsewhere (6). Forty-eight hours after incubation the plants were harvested. Only infected areas of leaves or comparable areas of control leaves were used. Twenty-five grams of fresh tissue were homogenized in 100 ml of acetone in a Virtis blender, the extract was filtered, and the residue was extracted twice more with 100-ml volumes of acetone. Additional acetone extracts contained no sterols. The acetone extracts were combined and the acetone was evaporated in vacuo, as was done in all other evaporation operations. The remaining aqueous extract was then made 33 I` with respect to acetone and extracted four times with 100-ml portions of diethyl ether. The ether phase was washed twice with 50-ml volumes of water, dried over Na2SO4 for 30 min, and evaporated to near dryness. Saponification was accomplished by addition of 50 ml of 95% ethanol and 5 ml of 60% KOH, followed by refluxing for 1 hr. After cooling, 200 ml of water were added and the mixture was then extracted thrice with 100-ml portions of CHCl3. The combined CHCl3 extract was washed twice with 50-ml volumes of water, dried over Na2SO4 for 30 min, evaporated to dryness, and dissolved in 2 ml of CHCl3. After adsorption on 0.5 g of alumina (activity III) the CHCI3 was evaporated. The alumina and adsorbed sample were applied to the top of a column (22