SOME FACTORS AFFECTING GROWTH OF APHANOMYCES EUTEICHES IN SYNTHETIC MEDIA

PAPAVIZAS, G. C., and C. B. DAVEY. (USDA, ARS, Crops Research Division, Beltsville, Maryland.) Some factors affecting growth of Aphanomyces euteiches in synthetic media. Amer. Jour. Bot. 47(9): 758-765. Illus. 1960.-Some nutritional requirements of 3 single-zoospore isolates of Aphanomyces euteiches Drechsler were studied in a synthetic medium (SM-1) consisting of mineral salts, D-glucose, DL-glutamic acid, and thioglycolic acid. Micronutrients were essential for growth, whereas vitamins were not. Controlled pH experiments showed that the fungus has a relatively wide pH range (5.2-7.7) for growth with an optimal range between 5.4 and 6.5. When the pH of the medium was maintained within the optimal range by periodic adjustments, ammonium nitrogen was utilized as the sole source of nitrogen. Nitrate nitrogen was unavailable to the fungus under all conditions. DL-glutamic acid was superior as a nitrogen source to either the D or the L isomer. Growth rate and final mycelial dry weight in a synthetic medium (SM-2) containing 14 amino acids in proportions found in 1.0% solution of yeast extract approximated those obtained in complex media. Mycelial dry weights in SM-2 were superior to those obtained in the glucose-glutamic acid-thioglycolic acid medium, but this superiority was due to DL-methionine, a sulfur-containing compound of the amino acid mixture. Sulfates were not utilized as sole source of sulfur, whereas cysteine, cystine, and thioglycolic acid supported fair amounts of growth. The optimal sulfur concentrations from thioglycolic acid for growth in SM-1 were between 128 and 192 mg/l. Mycelial yields equalling or exceeding those supported by complex media were obtained with high concentrations of D-glucose and DL-glutamic acid. GROWTH requirements of Aphanomyces, a member of the Saprolegniales, have received scant attention despite the fact that some species in the genus cause serious diseases of crop plants. Leonian and Lilly (1938) obtained good growth of A. camptostylus and of other water molds on synthetic media containing amino or ammonium nitrogen only when L-cystine was included. An unidentified species of Aphanomyces, among other saprolegniaceous fungi, made some growth on a glucose-alanine-cysteine medium (Volkonsky, 1933, 1934). A. stellatus and 4 other species of the Saprolegniales made good growth on a glucose-glutamic acid medium in the presence of cystine or other sources of reduced sulfur (Whiffen, 1945). Only a limited number of nutritional studies of Aphanomyces euteiches Drechsler, a serious root pathogen of peas, have been reported in the literature. Sherwood (1958) obtained some growth of the pea root pathogen on a modification of Whiffen's medium, but the growth rate on this synthetic medium was 1/3 of that supported by a yeast extract medium. No quantitative data were given in these studies. The fungus failed to grow on a synthetic medium with ammonium nitrogen as the sole source of nitrogen. Recently Haglund et al. (1959) reported growth of A. euteiches on a glucose-asparagine synthetic medium supplemented with methionine. No growth occurred in the medium with am1 Received for publication February 6, 1960. The authors are indebted to Dr. E. C. Cantino, Professor of Mycology, Michigan State University, East Lansing, Michigan, for reviewing this manuscript. Appreciation is also expressed to W. T. Schroeder for providing isolates 4 A 7 -1 +t UT A T-T-I.-A 1 C1 fnr -1_+_ ; C 7') monium or nitrate nitrogen substituting, for amino itrogen. Studies to elucidate some of the nutritional requirements of A. euteiches were undertaken by the authors in connection with a series of investigations on the biological control of the pea root-rot pathogen. This paper presents some of the results of these studies. MATERIALS AND METHODS.-Three single-zoospore isolates were used in stationary liquid cultures. Dry weight of mycelium was obtained by filtering the medium through tared filter paper in a Biichner funnel, washing with distilled water, drying the mycelium to constant weight at 820C., and weighing to the nearest 1 mg. on a Mettler single-pan electrical balance. Unless otherwise indicated, quintuplicate cultures were made in 40 ml. of medium contained in 250-ml. Erlenmeyer flasks covered with 2 layers of aluminum foil. Cultures were inoculated with disks cut from vigorously growing Petri-plate colonies by means of a biscuit cutter with an opening 4 mm. in diameter. The inoculum was grown on the basal medium used in each particular experiment. In all cases involving nitrogen, sulfur, and vitamins the inoculum was impoverished by 4 consecutive transfers on the basal medium lacking nitrogen, sulfur, and vitamins, respectively. Preliminary attempts to use zoospores or washed mycelial suspensions gave inconclusive and unreliable results. Unless otherwise indicated, cultures were incubated at 250C. for 20 days. All glassware was washed with detergent solution, concentrated chromic-sulfuric acid solution, and dilute hydrochloric acid. The glassware was rinsed