1997;50:117C127. factors that affect the development of sclerotia have been extensively reviewed previously (3, 16, 29, 30). Nutritional factors may stimulate (C, N, P, K+, Mg, S, and Zn2+) or inhibit (Al3+) development. Nonnutritional factors that influence sclerotial development include light, temperature, substrate pH, organic acid and stale product accumulation, phenolics, polyphenoloxidase activity, contact with mechanical barriers, -SH group modifiers, and osmotic potential. Although the list of factors known to influence sclerotial development is extensive, studies of these factors have been mostly observational. The underlying molecular mechanisms that regulate and signal this Eriodictyol development remain to be elucidated. We are interested in the molecular events that trigger and coordinate sclerotial morphogenesis. In recent years, signal transduction pathways linked to morphogenesis in phytopathogenic fungi have been studied for involvement in sporulation (8), spore germination (21, 28), appressorial development (13, 17, 28, 31C33), and filamentous or infectious growth (1, 4, 8, 10, 19, 20, 31). The genes and protein activities involved in these morphological processes include pheromone receptors (1), G-proteins (4, 19), mitogen-activated protein kinase (31), protein kinase A (17, 32, 33), and adenylate cyclase (10). Our objective was to examine the effects of various signal transduction effectors on sclerotial development to gain insight into which characterized signal transduction pathways are involved in sclerotial morphogenesis. MATERIALS AND METHODS Fungal isolates and growth conditions. The wild-type isolate of used in this study was isolate 1980 (ATCC 18683), obtained from dry bean culls in western Nebraska (9). In addition, 192 (ATCC 52585) (Canadian thistle, 1985, Montana), 222 (ATCC 18015) (sunflower, North Dakota, 1989), and 278 (ATCC 18687) (oil seed rape, Great Britain, 1995), 246 (ATCC 34327) (alfalfa, 1992), and 240 (ATCC 52583) (lettuce, 1969, New York) were provided by Jim Steadman (University of NebraskaLincoln). A single isolate, PR45 Ag-1-IB (ATCC 18619) (dry beans, Puerto Rico, 1995), was provided by Graciella Godoy (Ministry of Agriculture, Dominican Republic). Stocks of these isolates were stored as mycelia on desiccated paper discs or as sclerotia at ?20C. Fresh cultures were started from the paper disc stocks or sclerotia by sterile transfer onto potato dextrose agar (PDA) (Difco) plates. Activator and inhibitor studies. Cultures of isolate 1980 were grown on PDA supplemented with different concentrations of the following compounds known to affect conserved signal transduction pathways: staurosporine, H89, NaF, caffeine, KT5720, 3-isobutyl-1-methyl xanthine (IBMX), forskolin, diacyl glycerol kinase inhibitor I, okadaic acid, mastoparan, cholera toxin, verapamil, nifedipine, neodymium chloride, A23187, KN62, compound 48/80, and EGTA [ethylene-bis(oxyethylenenitrolo)tetraacetic acid]. When available, these compounds were obtained from Sigma Chemical Co. (St. Louis, Mo.). All other compounds except bPAK neodymium chloride and information concerning their modes of action were obtained from Calbiochem (San Diego, Calif.); neodymium chloride was purchased from Aldrich Chemical Co. (Milwaukee, Wis.). Cultures were grown in 2-cm-diameter wells of 24-well culture plates containing 2 ml of medium. Chemicals were added to the culture wells first and then thoroughly mixed with molten (45 to 50C) medium. Control cultures Eriodictyol were prepared in the same manner except that an equal volume of water or dimethyl sulfoxide was added depending on the solvent used to make the stock solution of each compound. After the medium had solidified, a mycelial plug (approximately 1 mm3) from a 5-day-old PDA culture was transferred to the center of each culture well. The cultures were incubated at room temperature (24 to.In general, vigorous mycelial growth precedes sclerotial development, with sclerotia produced when there is nutrient limitation (5). precedes sclerotial development, with sclerotia produced when there is nutrient limitation (5). Nutritional and environmental factors that affect the development of sclerotia have been extensively reviewed previously (3, 16, 29, 30). Nutritional factors may stimulate (C, N, P, K+, Mg, S, and Zn2+) or inhibit (Al3+) development. Nonnutritional factors that influence sclerotial development include light, temperature, substrate pH, organic acid and stale product deposition, phenolics, polyphenoloxidase activity, connection with mechanised obstacles, -SH group modifiers, and osmotic potential. However the list of elements known to impact sclerotial development is normally extensive, studies of the factors have already been mainly observational. The root molecular systems that regulate and sign this development stay to become elucidated. We want in the molecular occasions that cause and coordinate sclerotial morphogenesis. Lately, indication transduction pathways associated with morphogenesis in phytopathogenic fungi have already been studied for participation in sporulation (8), spore germination (21, 28), appressorial advancement (13, 17, 28, 31C33), and filamentous or infectious development (1, 4, 8, 10, 19, 20, 31). The genes and proteins activities involved with these morphological procedures consist of pheromone receptors (1), G-proteins (4, 19), mitogen-activated proteins kinase (31), proteins kinase A (17, 32, 33), and adenylate cyclase (10). Our objective was to examine the consequences of various indication transduction effectors on sclerotial advancement to gain understanding into which characterized indication transduction pathways get excited about sclerotial morphogenesis. Components AND Strategies Fungal isolates and development circumstances. The wild-type isolate of found in this research was isolate 1980 (ATCC 18683), extracted from dried out bean culls in traditional western Nebraska (9). Furthermore, 192 (ATCC 52585) (Canadian thistle, 1985, Montana), 222 (ATCC 18015) (sunflower, North Dakota, 1989), and 278 (ATCC 18687) (essential oil seed rape, THE UK, 1995), 246 (ATCC 34327) (alfalfa, 1992), and 240 (ATCC 52583) (lettuce, 1969, NY) were supplied by Jim Steadman (School of NebraskaLincoln). An individual isolate, PR45 Ag-1-IB (ATCC 18619) (dried out coffee beans, Puerto Rico, 1995), was supplied by Graciella Godoy (Ministry of Agriculture, Dominican Republic). Shares of the isolates were kept as mycelia on desiccated paper discs or as sclerotia at ?20C. Clean cultures were began in the paper disc stocks and shares or sclerotia by sterile transfer onto potato dextrose agar (PDA) (Difco) plates. Activator and inhibitor research. Civilizations of isolate 1980 had been grown up on PDA supplemented with different concentrations of the next compounds Eriodictyol recognized to have an effect on conserved indication transduction pathways: staurosporine, H89, NaF, caffeine, KT5720, 3-isobutyl-1-methyl xanthine (IBMX), forskolin, diacyl glycerol kinase inhibitor I, okadaic acidity, mastoparan, cholera toxin, verapamil, nifedipine, neodymium chloride, A23187, KN62, substance 48/80, and EGTA [ethylene-bis(oxyethylenenitrolo)tetraacetic acidity]. When obtainable, these compounds had been extracted from Sigma Chemical substance Co. (St. Louis, Mo.). All the substances except neodymium chloride and details concerning their settings of action had been extracted from Calbiochem (NORTH PARK, Calif.); neodymium chloride was bought from Aldrich Chemical substance Co. (Milwaukee, Wis.). Civilizations were grown up in 2-cm-diameter wells of 24-well lifestyle plates filled with 2 ml of moderate. Chemicals were put into the lifestyle wells first and thoroughly blended with molten (45 to 50C) moderate. Control cultures had been prepared very much the same except an equal level of drinking water or dimethyl sulfoxide was added with regards to the solvent utilized to help make the share solution of every compound. Following the moderate acquired solidified, a mycelial plug (around 1 mm3) from a 5-day-old PDA lifestyle was used in the center of every lifestyle well. The civilizations had been incubated at area heat range (24 to 26C) and examined for sclerotial advancement at seven days postinoculation. The consequences of cyclic AMP (cAMP) and 8-Br-cAMP had been evaluated very much the same. All treatments.