GROWTH AND SEXUAL REPRODUCTION OF APHANOMYCES EUTEICHES AS AFFECTED BY THE OXIDATION STATE OF SULFUR

DAVEY, C. B., and G. C. PAPAVIZAS. (Crops Res. Div., ARS, USDA, Beltsville, Md.) Growth and sexual reproduction of Aphanomyces euteiches as affected by the oxidation state of sulfur. Amer. Jour. Bot. 49(4): 400-404. Illus. 1962.-Growth and sexual reproduction of Aphanomyces euteiches Drechs., as affected by the oxidation state of sulfur (S), were studied in svnthetic media containing D-glucose, DiLglutamic acid, mineral salts, phosphate buffer, and various S sources. Organic and inorganic S sources representing some oxidation numbers (valences) were added to the media at concentrations to provide a range from 0 to 123 mg S/liter. The initial reaction in both liquid and solid media was adjusted to pH 6.2. Liquid cultures were maintained at that pH by periodic adjustment. Oxidized S with oxidation number +6 or +4 was not ultilized by the fungus, whereas good growth was obtained in the presence of S with oxidation numbers of 0 or -2. Although differences existed among isolates as to relative rates of utilization of the various S sources, there was complete conformity among isolates as to which sources were utilized for growth. Some isolates did not utilize all available S sources for sexual reproduction. EARLY STUDIES on vegetative growth in synthetic media of members of the genus Aphanomyces have been reported (Volkonsky, 1934; Leonian and Lilly, 1938; Whiffen, 1945). The growth and sexual reproduction of Aphanomyces euteiches Drechs., the incitant of a serious root rot of peas, had not until recently been the subject of studies employing chemically defined synthetic media (Papavizas and Davey, 1960a,b). In none of the work prior to that of the authors (Papavizas and Davey, 1960b) was sexual reproduction of the fungus observed on a synthetic medium. Studies on growth of A. euteiches have also been reported recently (Haglund, Durbin, and King, 1959; Haglund and King, 1960). All the foregoing investigations demonstrated that sulfate S was not available to Aphanomyces, whereas reduced S was. Cantino (1955) employed S utilization as one of the primary criteria in his phylogenetic scheme devised to explain the evolution of the aquatic Phycomycetes. The usual oxidation numbers of S are +6, +4, 0, and -2. In the present investigation oxidation number is used synonymously with valence and is a numerical expression of the oxidation state. The authors undertook a detailed studv of the use of S, in various oxidation states, by A. euteiches for both growth and sexual reproduction. The present report is the summation of these studies. 1 Received for publication September 15, 1961. The authors are indebted to Dr. C. E. Clapp for assistance with electrophoretic and chromatographic techniques. Appreciation is also expressed to Dr. WV. T. Schroeder for providing isolate 7, to Dr. W. A. Haglund for isolate 572, and to Mrs. L. 0. Lawyer for all isolates with prefix A. 2 For sake of brevity, sulfur is referred to as S throughout this paper. Where elemental S is intended it is indicated as S8 or elemental S. MATERIALS AND METHODS-Most of the mat erials and methods used were previously reported (Papavizas and Davey, 1960a,b). Briefly, the basal synthetic medium used was SM-1 (Papa vizas and Davey, 1960a) modified by increasing its carbon and nitrogen content. Carbon and nitrogen were provided as D-glucose (16.2 g/liter) and DL-glutamic acid * H20 (3.016 g/liter), respectively. The S source in SM-1, thioglycolic acid, was replaced by various S sources at several concentrations. The initial reaction in liquid and agar cultures was adjusted to pH 6.2 with 0.1 N NaOH or HCl before autoclaving and buffered at that value with phosphate3 buffer. Liquid media were placed in 250-ml Erlenmeyer flasks (40 ml/ flask), which were covered with 2 layers of aluminum foil. Stationary incubation of cultures was used throughout. The cultures were brought to the original pH every second or third day by aseptically adding sterile NaOH or HCl. The required volume of base or acid was determined by harvesting replicate flasks, determining the pH of the filtrates, and recording the volume of acid or base required to adjust the pH to the initial level. Twelve isolates of A. euteiches were used. Liquid and agar cultures were inoculated with uniform discs cut from Petri-plate colonies growing on the basal medium containing traces of S. Negligible amounts of S were transferred with the inoculum discs as inidicated by the minimal growth in control cultures. Unless otherwise indicated, cultures were incubated for 25 days at 25 C. Oospore production on the synthetic media solidified with Noble agar was quantitatively assayed after 15 days of incubation. Oospores I In ml/liter: M/3 KH2PO4, 15; M/3 Na2HPO4, 5.