VENTURIA INAEQUALIS (CKE.) WINT. V. THE INFLUENCE OF CARBON AND NITROGEN SOURCES AND VITAMINS ON GROWTH IN VITRO

STUDIES ON the inheritance of pathogenicity and of certain mutant characters of Venturia inaequalis (Cke.) Wint. with special reference to problems of parasitism and disease resistance (Keitt and Palmiter, 1938; Keitt and Langford, 1941; Keitt, et al., 1943; Shay and Keitt, 1945; Keitt et al., 1948) were initiated with the expectation that they would be supplemented by further investigations on the nutritional requirements of the pathogen. This paper reports comparative studies on the growth in vitro of certain pathogenic and nonpathogenic, mutant lines of the fungus in relation to various carbon and nitrogen sources and to certain vitamins. The chief purposes were to add to the background of knowledge of the nutrition of the fungus and to explore for information that might contribute to the elucidation of the nature of parasitism and disease resistance in apple scab. A preliminary report has been published (Leben, 1946). MATERIALS AND METHODS.-Lines of the fungus. -Seven lines of the fungus, certain of which had been used in the earlier studies, were employed in the present work. Data on the sources and characteristics of these lines follow. D8 is a wild-type, sporulating, pathogenic line that was derived from ascospore 8 of ascus D. This ascus was isolated in July, 1937, from an overwintered McIntosh apple leaf (Keitt and Langford, 1941). D8S arose in culture as a sector of D8. It is a nonpathogenic, sparsely sporulating line (Keitt and Langford, 1941) carrying the mutant gene, tan, which suppresses pathogenicity (Keitt et al., 1943). The tan locus is on the same chromosome arm as the locus for white (Shay and Keitt, 1945). On malt agar a D8S colony has a felty, tan surface. SD8S arose in culture as a sector of D8S. It is nonpathogenic and nonsporulating (Keitt and Langford, 1941). In addition to tan, SD8S carries the mutant gene, nonconidial, which suppresses or modifies pathogenicity (Keitt et al., 1943). Nonconidial has been located with reference to its centromere; it segregated independently from white and tan (Shay and Keitt, 1945). B 1 is a wild-type, sporulating pathogenic line derived from ascospore 1 of ascus B, which was isolated in July, 1937, from an overwintered McIntosh apple leaf (Keitt and Langford, 1941). BIS is a nonsporulating, nonpathogenic line that arose in culture as a sector of BI (Keitt and Lang1 Received for publication January 10, 1948. This work was supported in part by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation. Grateful acknowledgments are made to A. J. Riker for helpful counsel throughout the course of the investigation and to Eugene Herrling for taking the photographs. ford, 1941). Only four mature ascospores develop in each ascus when this line is crossed with compatible wild-type lines. 365-1 is a wild-type, sporulating, pathogenic line that grew from ascospore 1 of ascus 365, which was isolated from an overwintered Dudley apple leaf in February, 1942. This line was used in connection with studies on the inheritance of pathogenicity (Keitt et al., 1948). 454-2 is a wild-type, sporulating, pathogenic line derived from a single ascospore resulting from a cross in vitro in 1942 of two monoascosporic lines from ascus 0. Ascus 0 resulted from a cross in vitro of two monoascosporic lines from ascus B (v. s.). Line 454-2 was used in connection with studies on the inheritance of pathogenicity (Keitt et al., 1948). Stock cultures of the several lines of the fungus were grown at 160C. on bottle-slants of malt agar and transferred every 2 months by means of small fragments of mycelium. The lines were remarkably stable in culture, as attested by their uniform appearance and the consistent results of tests made at different times. They were tested for pathogenicity by the usual methods (Keitt and Langford, 1941) in August, 1945, and found to be pathogenic or nonpathogenic as recorded in 1941 and 1943. Cultural methods.-After trials with liquid and semi-solid media, an agar medium was selected as suitable for. the studies planned and was used for all the experiments that will be described. Distilled water and reagent grade chemicals were used. Glassware was cleaned in a potassium dichromatesulfuric acid solution and rinsed well with tap and distilled water. The pH determinations were made with a Beckman pH meter; for adjusting media, outside electrodes were employed. Adjustments were made with sodium hydroxide or sulfuric acid. The standard test vessels were pyrex test tubes, 25 X 100 mm., stoppered with cotton plugs covered with cheesecloth. The medium was dispensed in the tubes in 10 ml. amounts. Unless otherwise stated, sterilization was by autoclaving for 30 min. at 15 lb. pressure. Seeding.-Tubes were seeded by a mycelial fragment method developed for nonsporulating cultures. A flattenedneedle was employed to cut a portion about 5 X 2 X 2 mm. from the edge of a vigorously growing stock culture.2 This portion was transferred to a sterile Petri plate and on it was placed a piece of sterile glass.3 Pressure was ap2 For experiments reported in this paper, stock cultures that had grown approximately 1 month at 160C. were used. 3 It was found convenient to use a piece of microscope slide about 15 X 25 mm. It was autoclaved in a 25 X 100 mm. test tube and manipulated with flamed forceps.