Samples were taken by aseptically removing the mycelia growing within the cellophane and immediately submerging them in liquid nitrogen

Samples were taken by aseptically removing the mycelia growing within the cellophane and immediately submerging them in liquid nitrogen. nutrient limitation (5). Nutritional and environmental factors that impact the development of sclerotia have been extensively examined 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, temp, substrate pH, organic acid and stale product build up, phenolics, polyphenoloxidase activity, contact with mechanical barriers, -SH group modifiers, and osmotic potential. Even though list of factors known to influence sclerotial development is definitely extensive, studies of these factors have been mostly observational. The underlying molecular mechanisms that regulate and signal this development remain to be elucidated. We are interested in the molecular events that result in and coordinate sclerotial morphogenesis. In recent years, transmission 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 transmission transduction effectors on sclerotial development to gain insight into which characterized transmission 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), from dry bean culls in western Nebraska (9). In addition, 192 (ATCC 52585) (Canadian Haloperidol (Haldol) 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 or college of Haloperidol (Haldol) 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. New cultures were started from your paper disc shares or sclerotia by sterile transfer onto potato dextrose agar (PDA) (Difco) plates. Activator and inhibitor studies. Cultures of isolate 1980 were cultivated on PDA supplemented with different concentrations of the following compounds known to impact conserved transmission transduction pathways: staurosporine, H89, NaF, caffeine, KT5720, 3-isobutyl-1-methyl xanthine (IBMX), forskolin, diacyl glycerol kinase inhibitor Haloperidol (Haldol) 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 from Sigma Chemical Co. (St. Louis, Mo.). All other compounds except neodymium chloride and info concerning their modes of action were from Calbiochem (San Diego, Calif.); neodymium chloride was purchased from Aldrich Chemical Co. (Milwaukee, Wis.). Cultures were cultivated in 2-cm-diameter wells of 24-well tradition plates comprising 2 ml of medium. Chemicals were added to the tradition wells first and then thoroughly mixed with molten (45 to 50C) medium. Control cultures were prepared in the same manner except that an equal volume of water or dimethyl sulfoxide was NUFIP1 added depending on the solvent used to make the stock solution of each compound. After the medium experienced solidified, a mycelial plug (approximately 1 mm3) from a 5-day-old PDA tradition was transferred to the center of each tradition well. The cultures were incubated at space temp (24 to 26C) and then evaluated for sclerotial development at 7 days postinoculation. The effects of cyclic AMP (cAMP) and 8-Br-cAMP were evaluated in the same manner. All treatments and settings were set up in duplicate or triplicate. Treatments which affected sclerotial development in the primary screening were repeated a minimum of three times. cAMP assays. The cultures utilized for cAMP assays were set up in the same manner as the cultures utilized for inhibitor-activator studies, except that this medium surface was overlaid with cellophane before inoculation with the mycelial plug. The medium was supplemented with 5 mM caffeine for treatments or with an equal volume of water for controls. Cultures were produced for 3.