Purification and Partial Characterization of Host-Specific Toxins

WOLPERT, T. J., and L. D. DUNKLE. 1980. Purification and partial characterization of host-specific toxins produced by Periconia circinata. Phytopathology 70:872-876. Host-specific toxins isolated from culture filtrates of Periconia circinata properties of polyamines. HPLC resolved each of those toxic fractions into inhibited root growth of the susceptible sorghum genotype by 50% at 1 two compounds, a biologically active one and an inactive one. In addition to ng/ ml and had no effect on root growth of the near-isogenic resistant aspartic acid, both active compounds contained the same polyamine, and genotype at 2 Ag/ml. Purity of the isolated products was assessed by both inactive compounds contained another electrophoretically distinct thin-layer chromatography (TLC), thin-layer electrophoresis (TLE), and polyamine. The Periconia toxins are low-molecular-weight, acidic high-performance liquid chromatography (H PLC) of the toxins or their compounds containing multiple residues of aspartic acid and one or more dansyl derivatives. Two toxic substances were separated by preparative residues of a polyamine, which is responsible for ninhydrin reactivity and TLC; each contained only aspartic acid and two components with apparently responsible for selective biological activity. Additional key words: milo disease, Periconia root rot, Sorghum bicolor, selective pathotoxin. Pathogenicity of certain fungal pathogens is determined by hostsolution of the test material for 48 hr and evaluated for foliar specific toxins (8) (selective pathotoxins [13]), metabolic products wilting and necrosis (2). which, at low concentrations, affect only the hosts of the pathogen Root growth inhibition bioassays (2) were used to quantify toxin and produce the symptoms of the disease in the absence of the activity. Seeds were germinated at 25 C for 24 hr. Seedlings with pathogen. Thus, these toxins are assumed to elicit the biochemical roots 2-3 mm long were placed in 9-cm-diameter petri dishes responses of the host to the pathogen. The toxin-producing fungi containing 15 ml of 0.01 M KH 2 PO4 or solutions of toxin in 0.01 M and their hosts provide convenient systems for studies of hostKH 2 P0 4 and incubated in the dark at 25 C for 48 hr. To determine parasite interactions, because the mechanism of pathogenesis may activity, lengths of roots of susceptible and resistant seedlings be deduced from the mode of action of the toxin, incubated in test solution were compared with roots of seedlings Periconiacircinata (Mangin) Sacc., causal agent of milo disease incubated in 0.01 M KH 2PO 4. (4), produces a substance toxic only to genotypes of sorghum Toxin production. Single-spore isolates of P. circinata were (Sorghum bicolor [L.] Moench) that are susceptible to the obtained from roots of susceptible sorghum (6) and maintained on pathogen (11,12). For studies on the mechanisms of susceptibility potato-dextrose agar. For toxin production the fungus was grown and resistance, the toxin must be free of contaminating substances in standing culture at room temperature ('-24 C) for 10 days in that may induce responses not directly or necessarily involved in 400-ml prescription bottles containing 100 ml of modified-Fries' the initial stages of pathogenesis and in the host response to the medium (MF) supplemented with 0.1% yeast extract (7). After 10 pathogen. The objectives of this study were to develop a procedure days the medium was replaced with 100 ml of MF without yeast for purifying the toxin produced by P. circinata, to establish extract, and the cultures were incubated for an additional 15 days criteria for assessing the purity of the isolated product, and to (2). Removal of the yeast extract improved purification by describe some of the chemical properties of the toxin, eliminating several contaminating peptides and had no apparent effect on growth of the fungus. The culture filtrate (CF) was MATERIALS AND METHODS obtained by filtering the culture medium through four layers of cheese cloth. Plant material. Near-isogenic lines of Colby milo differing only Toxin purification. CF (2-6 L) was concentrated 20-fold in in the allelic form of the semidominant Pc gene for susceptibility to vacuo at 35 C. In all cases of in vacuo concentration, the sample was P. circinata (12) were used to assess toxin activity, cooled to 4 C until vacuum was established and again before Bioassays. A seedling bioassay (6) was used to determine toxic vacuum was released. The concentrated filtrate was allowed to activity of fractions during purification. Seedlings were grown in stand at 4 C for 24 hr. Insoluble material was removed by filtration, 25-ml beakers containing 20 ml of nutrient solution (5) and and the active supernatant was deproteinized by the addition of an incubated at room temperature ("•24 C) under continuous light equal volume of methanol and allowed to stand at 4 C for 24 hr. The (7,0001lx). Fiveto 7-day-old seedlings were incubated in 20 ml of a inactive precipitate was removed either by filtration or centrifugation. The methanol-soluble portion was concentrated 50-fold (with respect to original volume) to remove the methanol This article is in the public domain and not copyrightable. It may be freely analoetosndt4Cfr24h.Teoubeptinwshn reprinted with customary crediting of the source. The American Phytopathoadsorbed to activated charcoal (Norit A, Sigma Chemical Co., St. logical Society, 1980. Louis, MO 63178) (2.5 g/L of original CF) which was washed