The Mode of Action of Alternaric Acid on Myrothecium verrucaria

REILLY, C. C., and D. GOTTLIEB. 1979. The mode of action of alternaric acid on Myrothecium verrucaria. Phytopathology 69: 550-555. Alternaric acid inhibited the endogenous respiration of Myrothecium phosphorylation, were not affected by the antibiotic. Alternaric acid had no verrucaria but maximum effects of the toxin were reached after 5-6 min. noticeable effect on the activity of any enzyme of the Embden-MeyerhofThe toxin reduced the production of CO2 by 80-95% during aerobic Parnas pathway, the hexose monophosphate pathway, or the tricarboxylic respiration when (1 -C)-acetate, (2-C)acetate or (U"C)-glucose were acid cycle. Alternaric acid reduced the uptake of several common metabothe substrates. Alternaric acid totally inhibited the incorporation of 1"C lites by the mycelium, although not all to the same extent. The inhibition of from (UC)-glucose into the cell wall, protein, nucleic acid, lipid, and uptake occurred within the first 2 min. Thus, the mode of action of trichloroacetic acid-extractable cell fractions of the fungus. The toxin did alternaric acid appears to be the interference of uptake of metabolites by the not cause leakage of electrolytes from the mycelium of M. verrucaria. mycelium. Oxidation of exogenous substrates by isolated mitochondria, and oxidative Alternaria solani, the causal agent of early blight of tomato and produced by the cultures was extracted, purified, and characterized potato, produces a toxic metabolite, alternaric acid, in culture. by the methods of Grove (9). The compound thus obtained met the Brian et al (4) and Pound and Stahmann (13) demonstrated that criteria of melting point, UV absorption spectra, extinction coeffitreatment of tomato plants with alternaric acid caused lesions simicients, and biological activity for pure alternaric acid, and was used lar to those produced by the pathogen. The production of alternaric throughout this study. acid in culture varied with the isolate of pathogen, and there was no Myrothecium verrucaria (Alb. and Schw.) Ditm. ex Fries was correlation between pathogenicity and the production of the toxin obtained from the American Type Culture Collection, ATCC 9095, (4). Evidence for the production of the toxin in the host plant and was maintained on PDA. Submerged liquid cultures were during pathogenesis always was indirect and alternaric acid has not grown in Difco Czapek Dox Broth (CDB) modified by adjusting been demonstrated in the infected host. the pH to 3.6 with 1 N HCI prior to autoclaving. Portions (100 ml) Alternaric acid is selectively antifungal, inhibiting the growth of of CDB in 500-ml Erlenmeyer flasks were each seeded with 3 ml of some species of fungi at 1 Ag/ml or less, but not affecting other a suspension of 4-day-old mycelium which had been macerated in a fungal species even at 100 Aig/ml (3). Alternaric acid is not bacteriWaring Blendor. The cultures also were grown at 26 C on a reciprocidal (3), but is phytotoxic to many and diverse types of higher cal shaker for the periods required in the different experiments. plants (2). There is no information on the mechanism of action of Growth measurements. All fungal growth measurements were alternaric acid on fungi or higher plants, made on cultures grown in 50 ml of CDB in 250-ml Erlenmeyer The ultimate aim of our research was to determine the mode of flasks originally seeded with 1 ml of homogenized mycelium (2 mg action of alternaric acid on higher plants but the action of the toxin dry weight per milliliter). The average dry weight of the mycelium on Myrothecium verrucaria was first studied as a model for further was determined in triplicate and each experiment was repeated at investigations on higher plants. This paper describes the effects of least twice. alternaric acid on the growth and physiological processes of the The dry weights of cultures were measured at 12-hr intervals for mycelium of M. verrucaria. 6 days, At 4 days, the cultures were in late log phase and were used for subsequent experiments unless otherwise indicated. The toxic effect of ethanol, the solvent for alternaric acid, was determined at MATERIALS AND METHODS concentrations of 0.05 to 5% in the medium. Toxic action resulted Fungus cultures. An alternaric acid producing strain of in reduction of growth after 4 days and was determined by measurAlternaria solani (Ell. and G. Martin) L. R. Jones and Grant was ing the dry weight of mycelium. obtained from the American Type Culture Collection, ATCC The effects of alternaric acid on the growth of the fungus also was 11078. The liquid medium for the production of alternaric acid determined on 4-day-old mycelium by comparing the dry weight of consisted of: sucrose, 100 g; KNO3, 3.0 g; MgSO 4.7H 20, 0.5 g; cultures containing 0.5% ethanol to the cultures grown in the presKH 2PO4, 1.0 g; minor element concentrate, 1.0 ml; and distilled ence of the antibiotic. Alternaric acid in ethanol solution was added water to I L. The minor element concentrate consisted of: to triplicate flasks containing 50 ml of CDB at pH 3.5 to give FeSO,.7HO, 100 mg; MnSO 4.H 20, 10 mg; K2MoO 4.5HO, 10 mg; concentrations of 0.05 to 2.0 /g/ml and 0.5% (v/v), respectively. and distilled water to IL. The reaction of the medium was adjusted The effects of the pH of the biological activity of alternaric acid to pH 5.5-6.5 before autoclaving. Inoculum for the liquid cultures were measured in triplicate flasks containing 1 ttg/ml alternaric consisted of disks cut with a 5-mm diameter cork borer from the acid in 50 ml of CDB medium for each pH level. The control flasks edge of 10-day-old petri dish colonies of A. solani. Four disks were for each pH contained 0.5% ethanol. The effects of pH on the placed in each 500-ml Erlenmeyer flask containing 150 ml of the antibiotic were determined at initial pH levels ranging from 2.5 to liquid medium. The cultures were grown at 26 C for 15 days on a 7.0, after 4 days of growth. reciprocal shaker at 96 strokes per minute. The alternaric acid Effects of alternaric acid on respiration. Three-day-old liquid submerged cultures of M. verrucaria were harvested and suspended 00031-949X/79/000096$03.00/0 in 0.1 M sodium citrate, sodium phosphate buffer (CP buffer) pH @1979 The American Phytopathological Society 3.8 (6) (10 mg dry weight of mycelium per milliliter). Oxygen